Application of sr62 protein and its coding gene in resistance to wheat yellow mosaic virus

Abstract

This invention relates to the fields of plant molecular breeding, plant immunity, and plant disease detection, particularly the application of Sr62 protein and its encoding gene in resistance to wheat blast. This invention discovers a novel function of Sr62 in recognizing the wheat blast fungus effector protein PWT4, extending the known application of Sr62 in recognizing stem rust effector proteins to the fields of wheat blast effector protein recognition and resistance to wheat blast. Therefore, this invention provides a new technical foundation for expanding the application of Sr62 in wheat blast resistance and establishing a PWT4-specific detection system, and provides a new molecular tool for detecting avirulent genotypes of wheat blast fungus and assessing the risk of field strains. Simultaneously, this invention elucidates the Sr62-PWT4 recognition interface and the ATP-dependent complex formation conditions, providing a structural basis for screening Sr62 functional variants, engineering the recognition spectrum, and designing disease-resistant molecules, with broad application prospects.

Description

Technical Field

[0001] This invention relates to the fields of plant molecular breeding, plant immunity and plant disease detection, and in particular to the application of Sr62 protein and its encoding gene in resistance to wheat blast. Background Technology

[0002] Wheat blast is a major wheat disease caused by the wheat pathogen *Magnapordica oryzae*, which can cause panicle necrosis, grain shriveling, and yield loss. With the spread of the pathogen and changes in climate, the potential threat of wheat blast to wheat production continues to increase. Breeding and utilizing resistant varieties is one of the most economical and environmentally friendly strategies for controlling wheat blast; however, limited resistant gene resources, rapid differentiation of pathogen races, and the easy loss of resistance still seriously restrict the control of wheat blast.

[0003] Intracellular immune receptors in plant cells can recognize non-toxic effector proteins secreted by pathogens and initiate effector activation to trigger immunity. Tandem kinase proteins are an important class of immune receptors discovered in recent years in grasses, typically containing two tandem kinase domains. Wheat Sr62 was initially cloned as a stem rust resistance gene; its encoded protein, also known as WTK5, can recognize the stem rust effector protein AvrSr62 and activate the atypical NLR protein Sr62. NLR Or WTN1, which triggers an immune response. WTN1 and Sr62 NLR It has high sequence similarity and can be used as an executive immune protein downstream of Sr62.

[0004] PWT4 is an important non-toxic effector protein of wheat blast fungus. Existing research indicates that wheat tandem kinases Rwt4 or WTK3 can recognize PWT4 and induce an immune response through WTN1. Summary of the Invention

[0005] This invention is the first to discover that wheat tandem kinase Sr62 can not only recognize stem rust effector proteins, but also directly recognize wheat blast fungus effector protein PWT4 and activate WTN1-dependent ion channel activity, thereby triggering a plant immune response to enhance plant resistance to wheat blast. Based on this, the invention proposes the following technical solution.

[0006] In a first aspect, the present invention provides the use of Sr62 protein or its encoding gene, or biological materials containing Sr62 protein or its encoding gene, in the preparation of products for recognizing wheat blast fungus PWT4 and inducing plant immune responses.

[0007] In some implementations, the Sr62 protein recognizes wheat blast fungus PWT4 and activates the NLR executive protein, thereby inducing a plant immune response.

[0008] In some implementations, the Sr62 protein recognizes wheat blast fungus PWT4 and activates the NLR executive protein, thereby inducing ion channel activity and / or Ca2+. 2+ Related signals and / or hypersensitive cell death, thereby inducing plant immune responses.

[0009] In some embodiments, the NLR executive protein includes WTN1 or Sr62. NLR Or its functional homologs.

[0010] In some embodiments, the amino acid sequence of the Sr62 protein is any of the following: (1) As shown in SEQ ID NO.1; (2) An amino acid sequence with the same function as (1) obtained by substituting, deleting or adding at least one amino acid residue based on the amino acid sequence shown in (1); (3) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (1) or (2) (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%).

[0011] SEQ ID NO.1: MDGYEFERAEIDALERVVRDPREKPISLSFWLLRRITNDFSNASEIGRGGFSNVYLGVLPSGLRIAVKKLYQLTALANEFEDEVLTTMRVAHKNIVRLIGYCRHTQSEVFELKGESIFAQVRERLICTEYVPNGTLDGHITDKFHGGLDWDQRYQVLKGICHGLHYLHDEIRVIHRDIKTENILL DDNGVPKVFDFGFSKCLPDGESLIKDEATQGTLGYLAPESVDKQEYSFKSDIYSFGVVIIQLLTGSWISKNRNVELELKELRKRLVREGAFPSWENKYHQVRTCLEIVYNCMDIDPDKRPTALEIIQRLDETESANYSVSSGHATPLWQSGDEESDSAEVDAFEPERTPSKDILSDDEEPASADA NGETSTQKHNMPDLVSKQSVSAELSSLDFLEKITDGFSYEQILGKESLDADVHKAFLYEGSISGPAKVVVKRLIGVAIPVAKFKRDAEQLMSLDHKNIVKLLGYCHDESRGHKLVQFKAKPPQDFKGAEQLLCYEYMHNGSLREYLTGQGSREVDWHMCYKLIKGTCEGLRYLHEGCGDRPILHL NLNPSNILLDENNIPRITGFDFSKLIGEKNTKSVVLKKNGPLGYLPPDFLYSKGTDLKYLPTVDIYSLGLIILEIATRQEIKGDHGIIIRTVETNWRQDTQIATLYGSLEDKLREQVKMCIDIGLDCVKSNPKKRPTAGAIMLWLDKGSKPGVARPPLRDNVTHADDHIQEKEKQTPLRRFFRRK In some embodiments, the nucleotide sequence of the encoding gene is any one of the following: (1) As shown in SEQ ID NO.2; (2) A nucleotide sequence with the same function as (1) obtained by substituting, deleting or adding at least one nucleotide based on the nucleotide sequence shown in (1); (3) A nucleotide sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) homologous to the nucleotide sequence shown in (1) or (2).

[0012] SEQ ID NO.2: The biological materials include recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, plant cells or tissues.

[0013] In some implementations, the product is selected from any of the following: expression vector, plant cells or tissues, detection reagents or kits, and wheat blast fungus PWT4 effector protein detection model.

[0014] In a second aspect, the present invention provides the use of Sr62 protein or its encoding gene, or biological materials containing Sr62 protein or its encoding gene, in at least one of the following aspects: (1) Prepare transgenic plants resistant to wheat blast or wheat blast disease; (2) Enhance the plant’s resistance to wheat blast fungus or wheat blast disease or its immune recognition ability against wheat blast fungus; (3) Prepare products for improving plant resistance to wheat blast fungus or wheat blast disease resistance or wheat blast fungus immune recognition ability.

[0015] In some implementations, by introducing Sr62 protein or its encoding gene or biological material containing Sr62 protein or its encoding gene into plants, the plants express or overexpress Sr62 protein to obtain transgenic plants resistant to wheat blast fungus, or to enhance the plant's resistance to wheat blast fungus or its immune recognition ability against wheat blast fungus.

[0016] In some implementations, this is achieved by introducing the Sr62 protein or its encoding gene, or biological material containing the Sr62 protein or its encoding gene, into plants and co-expressing Sr62 and the NLR executive protein in the plants.

[0017] When plant material comes into contact with PWT4, Sr62 recognizes PWT4 and activates downstream NLR executive proteins, thereby inducing ion channel activity and Ca2+. 2+ Related signals or hypersensitive cell death can enhance plant resistance to wheat blast fungus or wheat blast disease, or improve the plant's immune recognition ability against wheat blast fungus.

[0018] In some implementations, the Sr62 protein in the plant recognizes PWT4 in wheat blast fungus and activates the NLR executive protein, thereby inducing ion channel activity and / or Ca2+. 2+ Related signals and / or hypersensitive cell death, thereby enhancing the plant's resistance to wheat blast fungus or its immune recognition ability against wheat blast.

[0019] In some implementations, enhancing plant resistance to or immune recognition of wheat blast fungus refers to enhancing plant resistance to or immune recognition of wheat blast fungus expressing PWT4.

[0020] In some implementations, the product is selected from any of the following: expression vector, plant cells or tissues, detection reagents or kits, and wheat blast fungus PWT4 effector protein detection model.

[0021] Thirdly, the present invention provides the use of Sr62 protein or its encoding gene, or biological materials containing Sr62 protein or its encoding gene, in at least one of the following aspects: (1) Screening for PWT4-type effector proteins; (2) Prepare a detection model for PWT4 effector protein of wheat blast bacteria.

[0022] In some implementations, the Sr62 protein or its encoding gene and the NLR executive protein or its encoding gene are co-expressed to obtain a wheat blast fungus PWT4 type effector protein detection model.

[0023] In some implementations, candidate PWT4 effector proteins or their encoding nucleic acids are co-expressed in the detection model with Sr62 protein or its encoding gene and NLR executive protein or its encoding gene to screen for PWT4 effector proteins.

[0024] In some implementations, the co-expressed system is a heterologous expression system.

[0025] Preferably, the heterologous expression system is selected from Xenopus laevis oocytes, Tobacco Benedict leaves, or wheat protoplasts.

[0026] In some implementations, the co-expressed system may also be a wheat system.

[0027] In some implementations, the detection model uses ion current or cell death as readout signals.

[0028] In some implementations, the generation of a readout signal indicates that the candidate PWT4 effector protein can be recognized by the Sr62 protein.

[0029] The PWT4 effector protein of this invention is a non-toxic effector protein secreted by the oat-derived isolate (Br58) of Pyricularia oryzae (Magnaportheoryzae). It can be specifically recognized by the wheat tandem kinase receptor WTK3 (Pm24) and activates the downstream immune response mediated by WTN1.

[0030] Fourthly, the present invention provides a method for screening PWT4 type effector proteins, comprising: co-expressing a candidate PWT4 type effector protein or its encoding nucleic acid with Sr62 protein or its encoding gene and NLR executive protein or its encoding gene, using ion current or cell death as readout signals, detecting the readout signals, and if a readout signal is generated, it indicates that the candidate PWT4 type effector protein can be recognized by Sr62 protein.

[0031] The method of this invention uses the Sr62-NLR effector protein as the core of the immune module and can screen for PWT4-type effector proteins that can be recognized by the Sr62 protein through membrane current or cell death. The screened PWT4-type effector proteins that can be recognized by the Sr62 protein can serve as new targets for preparing wheat blast-resistant plant materials or improving plant resistance to wheat blast.

[0032] Preferably, the NLR executive protein is selected from WTN1 or Sr62. NLR Or its functional homologs.

[0033] Preferably, the co-expression system is a heterologous expression system, more preferably an African Xenopus oocyte, Tobacco Benedict leaf, or wheat protoplast.

[0034] Fifthly, the present invention provides a method for detecting plant resistance to wheat blast fungus or wheat blast fungus immune recognition ability, comprising: determining plant resistance to wheat blast fungus or wheat blast disease resistance or wheat blast fungus immune recognition ability by detecting whether the plant contains the Sr62 and PWT4 complex.

[0035] In a specific implementation, the Sr62 and PWT4 complex in ATP and Mg 2+ It is formed under the condition of existence.

[0036] In a specific implementation plan, if the plant contains the Sr62 and PWT4 complex, it indicates that the plant has resistance to wheat blast fungus or wheat blast disease or the ability to recognize wheat blast fungus.

[0037] In a sixth aspect, the present invention provides the application of the method in the preparation of transgenic plants resistant to wheat blast or blast disease.

[0038] Preferably, the plant is a grass, more preferably wheat.

[0039] Seventhly, this invention elucidates the molecular mechanism by which Sr62 and PWT4 recognize each other, as detailed below: Further structural studies of this invention reveal that resting-state Sr62 forms symmetrical homodimers. Each Sr62 monomer contains two tandem kinase domains, Kin-1 and Kin-2. The β4–β5 region of Kin-1 forms an extended β-finger structure, and the Kin-1 β-fingers of the two monomers pair antiparallel and form a central groove; the corresponding region of Kin-2 forms a twisted anchoring structure and inserts into this groove, thereby maintaining the self-inhibitory state of Sr62.

[0040] Although PWT4 and the stem rust effector protein AvrSr62-5 differ significantly in sequence and topology, both can form a flexible biwing structure composed of two independent monolayer β-sheets. The biwing β-sheets of PWT4 embed into the Kin-1 β-finger grooves of the Sr62 dimer, forming a bimolecular clamp with the β-finger ends of the two Sr62 monomers and adjacent β3–αC rings. Upon binding, PWT4 competitively displaces the Kin-2 anchoring region, disrupting the self-inhibitory interface between Kin-1 and Kin-2, thus enabling Kin-2 to activate WTN1.

[0041] The Sr62-PWT4 recognition interface exhibits a multi-point synergistic characteristic. The A-wing of PWT4 contacts one Sr62 monomer via Tyr42, Ile43, Ile44, Thr45, Val50, and Val53, while the B-wing contacts another Sr62 monomer via Ile35, Tyr61, Gln62, Leu63, Ile64, Leu68, and Val71. Substitution of a single or few interface residues is usually insufficient to completely eliminate recognition, while simultaneously disrupting multiple key contact residues can significantly reduce or eliminate PWT4-induced Sr62-WTN1 activation. This flexible, multi-contact recognition mechanism explains Sr62's cross-pathogen recognition ability for different fungal effector proteins.

[0042] ATP binding can act as a regulatory step for the pre-activation of Sr62 conformation. The formation of a stable Sr62-PWT4 complex depends on ATP and Mg. 2+ However, no stable ATP retention was observed in the final complex, suggesting that ATP may transiently induce or stabilize a conformation that facilitates the entry of effector proteins into the Kin-1 groove, and then release them from the mature complex.

[0043] Compared with the prior art, the present invention has at least the following beneficial effects: This invention discovers a novel function of Sr62 in recognizing the wheat blast fungus effector protein PWT4, extending the known application of Sr62 in recognizing stem rust effector proteins to the fields of wheat blast effector protein recognition and resistance to wheat blast. Therefore, this invention provides a new technical foundation for expanding the application of Sr62 in wheat blast resistance and establishing a PWT4-specific detection system, and offers a new molecular tool for detecting non-virulent genotypes of wheat blast fungus and assessing the risk of field strains. Simultaneously, this invention elucidates the Sr62-PWT4 recognition interface and the ATP-dependent complex formation conditions, providing a structural basis for screening Sr62 functional variants, engineering the recognition spectrum, and designing disease-resistant molecules, demonstrating broad application prospects. Attached Figure Description

[0044] Figure 1 The results are based on electrophysiological detection of Xenopus laevis oocytes. Among them, A is the representative current trajectory recorded in the voltage range of -130mV to +70mV; B is the quantitative statistical result of the current amplitude at the clamp potential of -130mV; C and D are the representative curves of WTN1-mediated current and their quantitative statistical results of current amplitude after treatment with ion channel inhibitors.

[0045] Figure 2 This is based on the cell death detection results of tobacco leaves.

[0046] Figure 3 The results show the in vitro detection of the Sr62-PWT4 complex. A represents the elution profile of the Superose 6 10 / 300 GL molecular sieve, with the blue curve representing the Sr62 protein alone and the orange curve representing the Sr62-PWT4 co-incubated sample under ATP-free and MgCl2-free conditions. SDS-PAGE electrophoresis was performed on the eluted fractions. B shows the molecular sieve elution profile of the Sr62-PWT4 co-incubated sample in the presence of ATP and MgCl2, along with the corresponding SDS-PAGE electrophoresis results of the eluted fractions.

[0047] Figure 4 The results of the Sr62-PWT4 recognition mechanism analysis are as follows: A is the interaction interface structure diagram of Sr62-PWT4, in which the effector double-wing structure is inserted into the groove of the Sr62 dimer and captured by the Kin-1 gripper. The diagram shows the interaction residues on wings A, wings B and the Kin-1 gripper; B is the two-electrode voltage clamp detection results of the Kin-1-effector interface mutant and the quantitative statistics of the current amplitude at a clamp potential of -130mV; C is the cell death experiment results of different mutants in tobacco leaves. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0049] The following embodiments provide three independent detection schemes: electrophysiology, plant cell death, and in vitro complex formation. Those skilled in the art can select a single method or a combination of methods for verification according to experimental conditions, thereby improving the reliability of the detection.

[0050] In the embodiments provided in this specification, unless specific techniques or conditions are specified, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0051] This invention relates to molecular biology experiments. Unless otherwise specified, reference can be made to *Molecular Cloning: A Laboratory Manual, Fourth Edition*. This book is the most commonly used and guiding reference for those skilled in the art when performing experiments related to molecular biology. In addition, depending on the experimental purpose, those skilled in the art may conduct the corresponding experiments under the guidance of the operating manuals accompanying various commercially available kits or entrust the experiments to specialized companies.

[0052] Example 1: Electrophysiological detection results based on Xenopus laevis oocytes cRNAs of WTN1, Sr62, and the candidate PWT4 effector protein were prepared into solutions of approximately 50 ng / μL and mixed at a volume ratio of approximately 4:3:1. Positive controls used PWT4 and AvrSr62; negative controls used either a water-injected control or a combination lacking Sr62. Approximately 36.8 nL of the mixture was injected into each oocyte. Following injection, oocytes were incubated in ND96 buffer at approximately 18°C ​​for approximately 7 hours, and two-electrode voltage-clamp recordings (Warner brand) were performed 7 to 9 hours post-injection. ND96 buffer contained 96 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, and 5 mM HEPES, with a pH of approximately 7.6, and penicillin and streptomycin could be added.

[0053] During recording, the microelectrode was perfused with 3 M KCl, and the electrode resistance was preferably 0.5 to 1 MΩ. The test pulse was set from -130 mV to +70 mV in 20 mV steps, with each test pulse lasting approximately 6.6 seconds. The steady-state inward current at -130 mV was used as the preferred quantitative indicator. If a candidate PWT4 effector protein could induce a significantly enhanced inward current relative to the negative control, it was determined to be a PWT4 effector protein recognizable by Sr62. To avoid misjudgment due to differences in expression levels, the total amount of vector was kept consistent across groups, and at least three biological replicates were set up.

[0054] To further verify the relationship between current and Ca 2+ In relation to relevant immune channels, the calcium-activated chloride channel inhibitor CaCCinh-A01 or the calcium channel blocker LaCl3 were added. The PWT4-induced Sr62-WTN1-dependent current was expected to be partially inhibited by CaCCinh-A01 and completely or nearly completely inhibited by LaCl3. This pharmacological characteristic served as an auxiliary criterion for excluding nonspecific currents.

[0055] The amino acid and nucleotide sequences of the WTN1, Sr62, and candidate PWT4 effector proteins used in this embodiment are shown below: Sr62: The amino acid sequence is shown in SEQ ID No. 1, and the nucleotide sequence is shown in SEQ ID No. 2.

[0056] WTN1: The amino acid sequence is shown in SEQ ID No. 3, and the nucleotide sequence is shown in SEQ ID No. 4.

[0057] SEQ ID No. 3: SEQ ID No.4: PWT4: The amino acid sequence is shown in SEQ ID No. 5, and the nucleotide sequence is shown in SEQ ID No. 6.

[0058] SEQ ID No. 5: MRIQYFLPFLVADILAMSKPPSDSTDASKIPTYKIKHPGPRYIITDDGRVVEVERKESGRYQLIDGVLVRVEYEKKTGGSEGSKTSTNTRGPK SEQ ID No. 6: atgcgtattcaatattttcttccattcctcgtggctgatattttagctatgtccaaacccccctccgattcgacggatgcctcaaaaatcccgacttacaaaaattaaacatccagggcctcgttacattataactgacgat ggtcgagttgttgaggtcgaacgcaaggaatcaggacgctatcaattaatcgatggggttttagtacgtgtggaatatgaaaagaaaaccgggggttcggaagggtcaaaaacatccaccaacacgcgcggtccgaaataa PWT4 Br48 The amino acid sequence is shown in SEQ ID No. 7, and the nucleotide sequence is shown in SEQ ID No. 8. It is approximately 80% homologous to PWT4.

[0059] SEQ ID No.7: MRIQYFLPSLVAGILAMPKPSSNSLNSPEVPTYKIGHPGPRYIRYNDGEIHEVERKESGRYRVDDGGLIREEYEKKTQDSEGSKTPTTRGPK SEQ ID No. 8: atgcgtatccaatattttcttccatccctcgtggctggtattttagccatgcccaaaccctcctccaattcgctgaattccccagaagtcccgacttataaaattggacatcctgggcctcggtatattagatataacgatggtgaaatccatgaggtcgaacgcaaggaatcagggcgctatcgagtagacgacgggggtttaatacgtgaggaatatgaaaaaaaaactcaagattcggaagggtcaaaaactcccaccacgcgcggtcctaaataa AvrSr62-5: the amino acid sequence of which is set forth in SEQ ID No.9, and the nucleotide sequence of which is set forth in SEQ ID No.10.

[0060] SEQ ID No.9: MLLKSLCMAVYALNHCMFSSSLAKPVMKRSIERFGMEGSSSVEESPFHGPLYESNEYHNINGDLHIVSWYCTPPEITFHFNGKDRKVTYHEFLMLGDSDGISKSDKQAMTNALISKEQGSKVIGMSQIGNQPISAEEKNRKWPNGPPRFIKTTDMIHSNTDGRKIVYRYFRTTPVAEKI SEQ ID No.10: atgttgttgaaatcactctgcatggcagtttatgccttaaatcactgtatgttttcatcttcactggcaaagcctgttatgaagagatcaatcgaaaggtttgggatggagggaagttcgtctgttgaagaatctccttttcacggacctttatatgaatccaatgagtaccataacatcaatggggatctgcatattgttagctggtactgtacaccccctgaaataacattccacttcaatggaaaagataggaaagttacataccatgagtttttaatgcttggtgactcagatgggataagtaaatctgacaaacaggcaatgacaaatgcattgatatccaaagagcaaggttccaaagttattggtatgagtcaaattggaaatcaaccaatctcagccgaggaaaagaatagaaaatggcccaatgggcctccacgctttatcaagacaacagatatgattcattctaatacagatggcagaaaaattgtttacagatacttccgtacaactcctgttgcagagaaaatttaa AvrSr62-7: the amino acid sequence is shown in SEQ ID No.11, and the nucleotide sequence is shown in SEQ ID No.12.

[0061] SEQ ID No.11: MLLKSILMMFCIFYHCMLPESFAKPIMKSSIFKGGETSSLGESPFEGPLYKYEEIVHAQGKFLVTTYYRTPIKLEFPFNGKDISISLNEFSKNSDSLGISKEEKEKVTKTINSEGNGAQSSTMGQNKGKELSIEEKEKIWPDGPPRFDEKKYGIKLRDANGDRTIFYKYSHTPYVVTELNLNP SEQ ID No.12: atgttactgaaatcaatcttgatgatgttttgcatcttctatcactgtatgcttccagagtcttttgcaaaacctataatgaaaagctccatttttaaaggaggggaaacttcatctcttggagaatccccttttgaa ggaccgttgtacaaatatgaagaaatagtgcatgcccaggggaagtttttggtcactacctattatcgtacacccataaaattagaatttcctttcaatggaaaagatatctccatatcattaaatgaattttcaaag aacagtgattcattgggaatcagtaaagaagaaaaagagaaagtcaccaagacaattaactctgaaggaaatggtgctcaaagttctacaatgggccaaaataagggaaaagagctttccattgaggaaaaggaaaaa atttggcctgatggtccccacgctttgatgagaagaaatatggaattaaattacgcgatgccaatggtgacagaacaatattctacaaatattcccataccccttacgttgttacagagctcaacttaaatccttga Test results are as follows Figure 1 As shown, the results indicate that Sr62 can recognize effector proteins AvrSr62-5, AvrSr62-7 and PWT4, and initiate downstream signaling to induce WTN1-mediated ion flow.

[0062] Example 2: Cell death detection based on tobacco leaves WTN1, Sr62, and candidate effector proteins (AvrSr62-5, AvrSr62-7, and PWT4) were constructed into the plant expression vector pC1307. WTN1 carried a Strep-Flag-Strep tag at its C-terminus, Sr62 and its mutants carried an HA tag at their N-terminus, and the candidate effector proteins were untagged. The vectors were then transformed into Agrobacterium GV3101. Agrobacterium was cultured overnight at 28°C and 200 rpm in LB medium containing 50 μg / ml Kanamycin and 25 μg / mL Rifampicin. After centrifugation at 6000 rpm for 1 min, the precipitate was resuspended in an infiltration buffer containing 10 mM MES (pH=5.6), 10 mM MgCl2, and 200 μM acetylsyleugenone. The OD values ​​of the WTN1 bacterial culture were then calculated.600 Adjust to approximately 0.1, Sr62 bacterial culture OD 600 Adjusted to approximately 0.25, OD of effector protein bacterial culture 600 Adjust to approximately 0.3, mix the three ingredients in equal volumes, and let stand in the dark at room temperature for about 2 hours. Use a needle-free syringe to soak the fully expanded leaves of 3–4 week old Nicotiana benthamiana.

[0063] Three to five days after infiltration, hypersensitive cell death was observed and recorded under white and ultraviolet light. If co-expression of the effector protein with Sr62 and WTN1 resulted in significant and reproducible cell death, while the corresponding negative control did not produce cell death, it indicates that the candidate effector protein can trigger a Sr62 / WTN1-mediated immune response, suggesting that it may be recognized by Sr62. This result can be cross-validated with the oocyte electrophysiological results from Example 1.

[0064] Test results are as follows Figure 2 As shown in the figure. The results indicate that Sr62 can recognize effectors AvrSr62-5, AvrSr62-7 and PWT4, and induce hypersensitivity responses in Benedictine leaves.

[0065] Example 3: Detection of the Sr62-PWT4 complex in vitro Sr62 was expressed and purified in the HEK293F mammalian cell expression system. The candidate effector protein was expressed and purified in *E. coli*, and the predicted signal peptide was removed. The purified Sr62 and the candidate effector protein were mixed at a molar ratio of approximately 1:5 and incubated overnight at approximately 4°C in the presence of approximately 10 mM ATP and approximately 10 mM MgCl2. The complex was then separated using a Superose 6 size exclusion column, and the co-migration of Sr62 and the candidate effector protein was confirmed by SDS-PAGE, Western blotting, or mass spectrometry. Buffer: 25 mM Tris pH 8.0; 150 mM NaCl; 2 mM DTT; 0.5 mM TCEP.

[0066] Test results are as follows Figure 3 As shown, under the same conditions but without the addition of ATP, Sr62 and PWT4 cannot form a stable co-migration complex; however, with the addition of ATP and Mg... 2+ Subsequently, the two can form a stable complex. Therefore, ATP / Mg 2+ The appearance of the dependent co-migration peak serves as a biochemical indicator of the specific binding of Sr62 to candidate effector proteins.

[0067] Therefore, the presence of the Sr62 and PWT4 complex in a plant can be used to determine its resistance to wheat blast fungus, its resistance to wheat blast disease, or its immune recognition ability against wheat blast. The presence of the Sr62 and PWT4 complex in a plant indicates that it possesses resistance to wheat blast fungus, its resistance to wheat blast disease, or its immune recognition ability against wheat blast.

[0068] Example 4: Analysis of the Recognition Function of Sr62-PWT4 This embodiment, through structural analysis of the Sr62-PWT4 complex, reveals that PWT4 is embedded in the Kin-1 β-finger grooves of the Sr62 dimer via biwing β-sheets. The A-wing of PWT4 contacts one side of the Sr62 monomer, with key residues including Ile35, Tyr61, Gln62, Leu63, Ile64, Leu68, and Val71; the B-wing contacts the other side of the monomer, with key residues including Tyr42, Ile43, Ile44, Thr45, Val50, and Val53. The Sr62 side interface residues include Gln72, Leu73, Leu76, Phe110, Leu112, Glu115, and Ile117. Figure 4 ).

[0069] Further functional verification through mutation of key residues showed that the PWT4 interface has a certain tolerance to local substitution, while the A-wing four mutation Y42A / I43A / I44A / V50A or the B-wing five mutation Y61A / Q62A / L63A / I64A / L68A can significantly reduce or eliminate Sr62 recognition and WTN1 activation. Figure 4 These results suggest that PWT4 escaping Sr62 recognition requires synergistic alterations at multiple key contact sites, and this invention provides a molecular basis for assessing the potential immune escape risk of field strains.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of Sr62 protein or its encoding gene, or biological materials containing Sr62 protein or its encoding gene, in the preparation of products for recognizing wheat blast fungus PWT4 and inducing plant immune responses.

2. The use of Sr62 protein or its encoding gene, or biological materials containing Sr62 protein or its encoding gene, in at least one of the following aspects: (1) Prepare transgenic plants resistant to wheat blast or wheat blast disease; (2) Enhance the plant’s resistance to wheat blast fungus or wheat blast disease or its immune recognition ability against wheat blast fungus; (3) Prepare products for improving plant resistance to wheat blast fungus or wheat blast disease resistance or wheat blast fungus immune recognition ability.

3. The use of Sr62 protein or its encoding gene, or biological materials containing Sr62 protein or its encoding gene, in at least one of the following aspects: (1) Screening for PWT4-type effector proteins; (2) Prepare a detection model for PWT4 effector protein of wheat blast bacteria.

4. A method for screening PWT4 type effector proteins, characterized in that, include: Candidate PWT4 effector proteins or their encoding nucleic acids are co-expressed with Sr62 protein or its encoding gene and NLR executive protein or its encoding gene. Ion current or cell death is used as readout signals. If a readout signal is generated, it indicates that the candidate PWT4 effector protein can be recognized by Sr62 protein.

5. The method according to claim 4, characterized in that, The NLR executioner protein is selected from WTN1 or Sr62 NLR or a functional homologue thereof.

6. The method according to claim 4 or 5, characterized in that, The co-expression system is a heterologous expression system, preferably Xenopus laevis oocytes, Tobacco Benedict leaves, or wheat protoplasts.

7. A method for detecting plant resistance to wheat blast fungus or its immune recognition ability, characterized in that, include: The presence of the Sr62 and PWT4 complex in plants can be used to determine plant resistance to wheat blast fungus, wheat blast disease resistance, or the ability to recognize wheat blast fungus.

8. The method according to claim 7, characterized in that, The Sr62and PWT4 complex forms in the presence of ATP and Mg 2+ under the presence of ATP and Mg2+.

9. The method according to claim 7 or 8, characterized in that, The presence of the Sr62 and PWT4 complex in a plant indicates that the plant possesses resistance to wheat blast fungus, or resistance to wheat blast disease, or the ability to recognize wheat blast fungus.

10. The use of the method according to any one of claims 7 to 9 in the preparation of transgenic plants resistant to wheat blast or blast disease.

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