Application of CPKs-TIR-NLRs pathway in regulation of plant immunity

Abstract

The invention relates to an application of a CPKs-TIR-NLRs pathway in regulation of plant immunity. The invention provides a method for regulating the immunocompetence of a plant. The method comprises the step of regulating a CPKs-TIR-NLRs signal channel or a channel molecule thereof in the plant. The CPKs disclosed by the invention can phosphorylate TIR-NLR, so that the immunity of plants is regulated, and the growth of the plants is promoted. The invention has important significance on genetic improvement of plant traits.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and botany, and more specifically, the present invention relates to the application of CPKs-TIR-NLRs pathway in regulating plant immunity. Background Art

[0002] Plants face multiple challenges in their survival and growth in nature, including biotic stress, abiotic stress, and plant-to-plant competition. Plants activate immune and stress signals to survive under a variety of stresses, but this also results in a lack of sufficient competitiveness at the expense of inhibiting plant growth, especially in plant-to-plant competition under normal growth conditions. Balancing and finely regulating the relationship between plant growth and stress response will enable them to survive and thrive in a complex and changing natural environment. Plants have evolved a variety of specific and shared signaling pathways to respond to stress environments. The art does not yet have a deep understanding of the fine regulation that occurs in plants.

[0003] In order to better resist stress or pathogen invasion, plants have evolved two different immune systems, PTI (pattern-triggered immunity) and ETI (effector-triggered immunity). Among them, ETI triggers plant immunity by recognizing effector proteins secreted by pathogens into cells through NLR (Nucleotide-binding Leucine-rich Repeat) receptors that bind to nucleotides and are rich in leucine repeat sequences in cells. After the NLR receptor protein in the cell recognizes the effector protein, it triggers the plant's ETI immune signal, and the ETI immune signal causes programmed cell death (PCD) in the local part of the plant, thereby protecting the plant as a whole from pathogen infection. According to the different domains at the N-terminus, NLR proteins are divided into TNL-type resistance (R) proteins with TIR (Toll / Interleukin-1 receptor / Resistance) domains at the N-terminus and CNL-type resistance (R) proteins with CC (coiled-coil) domains at the N-terminus.

[0004] Osmotic stress and pathogen invasion can activate Ca 2+ Signaling activity and NLR protein signals containing a TIR domain at the N-terminus, such as SNC1 and ACQOS / VICTR. TNL-type R proteins are typical plant intracellular immune receptors that contain an N-terminal TIR domain and can hydrolyze NAD + and NADP +and consume DNA or RNA to produce 2′,3′-cAMP / cGMP; NB-ARC domain, responsible for oligomeric proteins; and LRR domain, involved in recognizing pathogen effector factors and having autoinhibitory protein activity functions. Although TIR-NLR type R proteins can improve plant resistance to pathogens, they can also antagonize plant tolerance to ABA-dependent hyperosmotic stress and plant growth. Therefore, how to finely regulate the balance between TIR-NLR signals and plant growth and tolerance to osmotic stress is a scientific problem that needs to be solved urgently. Summary of the invention

[0005] After in-depth research, the inventors provide a method for regulating the immunity of plants (such as Arabidopsis), comprising: regulating the CPKs-TIR-NLRs signaling pathway or its pathway molecules in the plant. The present invention reveals that CPKs can phosphorylate TIR-NLR, thereby regulating the immunity of plants (such as Arabidopsis), thereby promoting plant growth. The present invention is of great significance for the genetic improvement of plant traits.

[0006] In a first aspect of the present invention, a method for regulating plant immunity is provided, comprising: regulating the CPKs-TIR-NLRs signaling pathway or its pathway molecules in the plant.

[0007] In one or more embodiments, plant immunity is regulated by regulating CPKs-TIR-NLRs or their pathway molecules (promoting / activating the interaction of pathway molecules); the regulation of plant immunity includes: enhancing (including maintaining) the plant's ability to respond to stress or its tolerance to stress, inhibiting plant cell death, and promoting (including maintaining) plant growth.

[0008] In one or more embodiments, the regulation of the CPKs-TIR-NLRs signaling pathway or its pathway molecules includes: increasing the phosphorylation level of TIR, promoting CPKs-TIR interaction to inhibit TIR, and regulating the immune capacity of the plant; preferably, increasing the phosphorylation level of TIR includes: increasing the phosphorylation level of Ser at position 26 and / or position 18 of TIR; preferably, simulating phosphorylation modification by mutating Ser to Asp.

[0009] In one or more embodiments, the mutation of Ser at position 26 and / or position 18 to Asp (abbreviated as S26D, S18D) means that the amino acid residue at position 26 and / or position 18 of the protein encoded by SNC1 is mutated from S to D.

[0010] In one or more embodiments, the regulation includes: enhancing the ability of CPKs to phosphorylate TIR; transferring the coding gene of CPKs or an expression construct or vector containing the coding gene into the plant; activating CPKs with calcium ions (including calcium salts that can produce calcium ions); performing gain-of-function mutations on CPKs; promoting the expression of CPKs with an expression-enhancing promoter or a tissue-specific promoter; or promoting the expression of CPKs with an enhancer.

[0011] In one or more embodiments, the CPKs include CPK3, CPK4, CPK5, CPK6, and CPK11.

[0012] In one or more embodiments, the TIR includes: a SNC1 protein, a TNLs protein or a NLRs protein containing a TIR domain.

[0013] In a second aspect of the present invention, there is provided the use of the CPKs-TIR-NLRs signaling pathway, its pathway molecules or their regulators for regulating the immune capacity of plants.

[0014] In one or more embodiments, the CPKs include CPK3, CPK4, CPK5, CPK6 and / or CPK11.

[0015] In one or more embodiments, the regulator is used to regulate plant immunity; the regulation of plant immunity includes: enhancing (including maintaining) the plant's ability to respond to stress or its tolerance to stress, inhibiting (including maintaining) plant cell death, and promoting (including maintaining) plant growth.

[0016] In one or more embodiments, the regulator includes: an agent that increases the phosphorylation level of TIR, and an agent that promotes CPKs-TIR interaction and thereby inhibits TIR; preferably, the agent that increases the phosphorylation level of TIR includes: an agent that increases the phosphorylation level of Ser at position 26 and / or position 18 of TIR, and more preferably includes an agent that mutates Ser to Asp (such as a point mutation agent, more specifically a point mutation construct / carrier material).

[0017] In one or more embodiments, the regulator includes: an exogenous CPKs encoding gene or an expression construct or vector containing the encoding gene, calcium ions (including calcium salts that can produce calcium ions), or an agent for performing gain-of-function point mutations on CPKs.

[0018] In one or more embodiments, the CPKs include CPK3, CPK4, CPK5, CPK6 and / or CPK11.

[0019] In one or more embodiments, the stress comprises osmotic stress (high osmotic stress); preferably, the osmotic stress comprises abiotic stress; preferably, the abiotic stress comprises: salt (high salt) stress, cold stress, drought stress.

[0020] In one or more embodiments, the regulation includes up-regulation or down-regulation.

[0021] In one or more embodiments, the upregulation, promotion, increase or enhancement indicates a significant upregulation, promotion, increase or enhancement, such as upregulation, promotion, increase or enhancement by 5%, 10%, 15%, 20%, 40%, 60%, 80%, 90% or more.

[0022] In one or more embodiments, the downregulation, reduction, inhibition, weakening or attenuation means significant downregulation, reduction, inhibition or weakening, such as downregulation, reduction, inhibition or weakening by 5%, 10%, 15%, 20%, 40%, 60%, 80%, 90% or lower.

[0023] In a third aspect of the present invention, there is provided a method for identifying plant immunity, comprising: analyzing the accumulation level of phosphorylated proteins of the plant's TIR domain and the level of protein phosphorylation;

[0024] If the accumulation level of phosphorylated proteins in the TIR domain and / or the protein phosphorylation level is normal (or high), the plant immune capacity is normal (or low); if the accumulation level of phosphorylated proteins in the TIR domain and / or the protein phosphorylation level is abnormal (including disorder or down-regulation), the plant immune capacity is abnormal (or high).

[0025] In one or more embodiments, analyzing the phosphorylation level of a protein includes analyzing the phosphorylation level of Ser at position 26 and / or Ser 18 of TIR.

[0026] In one or more embodiments, the plant immunity includes: the ability to respond to osmotic stress or the ability to tolerate stress, the survival of plant cells, and the growth of plants.

[0027] In a fourth aspect of the present invention, a method for screening a substance (potential substance) for regulating plant immunity is provided, comprising:

[0028] (1) adding the candidate substance to a system expressing the CPKs-TIR-NLRs signaling pathway or its pathway molecules;

[0029] (2) detecting the system and observing the phosphorylation level of TIR therein; if the phosphorylation level is increased, it indicates that the candidate substance is a substance that can be used to regulate plant immunity.

[0030] In one or more embodiments, increasing the phosphorylation level of TIR includes: increasing the phosphorylation level of Ser at position 26 and / or Ser at position 18 of TIR.

[0031] In one or more embodiments, the CPKs include CPK3, CPK4, CPK5, CPK6 and / or CPK11.

[0032] In one or more embodiments, the TIR includes: a SNC1 protein, a TNLs protein or a NLRs protein containing a TIR domain.

[0033] In one or more embodiments, (2) includes: observing the effect of the candidate substance on the interaction between CPKs and TIR; if it promotes the interaction between the two and promotes CPKs to phosphorylate TIR, it indicates that the candidate substance is a substance that can be used to regulate plant immunity; preferably, the CPKs phosphorylation of TIR includes: phosphorylating Ser at position 26 and / or position 18 of TIR.

[0034] In one or more embodiments, a control group is also included to clearly distinguish the difference in expression, activity or phosphorylation level of CPKs, CPKs-TIR-NLRs or CPKs-TIR signaling pathway proteins in the test group and the control group.

[0035] In one or more embodiments, the candidate substances include (but are not limited to): regulatory molecules (such as upregulators, downregulators, mutagenesis agents, small molecule compounds, gene editing constructs, etc.) designed for CPKs, TIR domains, SNC1, TNLs or NLRs containing TIR domains or their encoding genes or their upstream or downstream proteins or genes.

[0036] In a fifth aspect of the present invention, there is provided a use of a plant CPKs-TIR-NLRs signaling pathway or a molecule in its pathway as a molecular marker for identifying the immune capacity of a plant.

[0037] In one or more embodiments, the CPKs include CPK3, CPK4, CPK5, CPK6 and / or CPK11.

[0038] In one or more embodiments, the plant comprises a plant selected from the group consisting of: a plant containing a CPKs-TIR-NLRs signaling pathway; a dicotyledonous plant; a flax plant; and a cruciferous plant.

[0039] In one or more embodiments, the dicotyledonous plants include (but are not limited to): soybean; the flax plants include (but are not limited to): flax; the cruciferous plants include (but are not limited to): Arabidopsis thaliana, cabbage, kale, mustard, radish, and rapeseed.

[0040] In one or more embodiments, the proteins or genes in the CPKs-TIR-NLRs signaling pathway include their homologs.

[0041] In one or more embodiments, the CPK3 protein comprises a protein selected from the group consisting of:

[0042] (a1) a protein having an amino acid sequence as defined in SEQ ID NO:1; (b1) a protein derived from (a1) having the function of the protein defined in (a1) and formed by substitution, deletion or addition of one or more (e.g., 1-20; preferably, 1-15; more preferably, 1-10, such as, 5, 3) amino acid residues of the amino acid sequence of SEQ ID NO:1; (c1) a protein derived from (a1) having more than 80% (preferably, more than 85%; more preferably, more than 90%; more preferably, more than 95%, such as, 98%, 99%) homology with the protein sequence defined in (a1) and having the function of the protein defined in (a1); or (d1) a protein active fragment defined in (a1), or a protein formed by adding a tag sequence, a restriction enzyme sequence or a reporter protein to both ends of the protein; and / or

[0043] (a2) a protein having an amino acid sequence such as SEQ ID NO:2; (b2) a protein derived from (a2) having the function of the protein (a2) and obtained by replacing, deleting or adding one or more (such as 1-20; preferably 1-15; more preferably 1-10, such as 5, 3) amino acid residues of the amino acid sequence of SEQ ID NO:2; (c2) a protein derived from (a2) having more than 80% (preferably more than 85%; more preferably more than 90%; more preferably more than 95%, such as 98%, 99%) homology with the protein sequence defined in (a2) and having the function of the protein (a2); or (d2) a protein active fragment defined in (a2), or a protein formed by adding a tag sequence, an enzyme cleavage sequence or a reporter protein to both ends of the protein.

[0044] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The dominant negative mutation of SNC1 can inhibit NLR signaling to maintain plant growth. (a) The location of the mutated SNC1 site in the ios mutant on its sequence. (b) AEQsig6, bon1, ios22 and OE-SNC1 G1343D The rosette leaf growth phenotype of the transgenic material of AEQsig6, bon1, ios22 and OE-SNC1 15 days after treatment with 100 mM mannitol. Scale bar, 0.5 cm. (c) AEQsig6, bon1, ios22 and OE-SNC1 G1343D(d) Growth phenotype of AEQsig6, bon1-7 and ios26 mutants after 15 days of treatment with 100 mM mannitol. Scale bar, 0.5 cm. (e) Growth phenotype of ios26, bon1 and OE-SNC1 V518I Growth phenotype of / bon1 transgenic material after 5 weeks of growth in soil. Scale bar, 2 cm.

[0046] Figure 2 The osmotic stress-induced increase in PR1 / PR2 and SA accumulation in the bon1 mutant were restored by the ios mutant. G1343D Relative expression of PR1 (a) and PR2 (b) and SA (c) content in vivo (unit: ng / g fresh weight) of transgenic materials of / bon1-7 after 5 days of treatment with 100 mM mannitol. Values ​​are standard deviation ± SD. (a) and (b), n = 3.

[0047] Figure 3 snc1-1 has a typical osmotic stress sensitive phenotype. Rosette leaf growth phenotypes of Col-0 and snc1-1 mutants 15 days after 100 mM mannitol treatment. Scale bar, 0.5 cm.

[0048] Figure 4 Osmotic stress activates the kinase activity of CPK3. (a) Measurement of calcium signaling in vivo in bon1-7 and ios102 mutants after treatment with 600 mM mannitol. (b) Expression of CPK3, CPK4, CPK5, CPK6, and CPK11 in Arabidopsis. (c) Osmotic stress activates the kinase activity of CPK3.

[0049] Figure 5 , CPK3CA inhibits NLR signaling to regulate plant growth. Note: (a) Rosette leaf growth phenotype of Col-0, bon1 and CPK3pro::CPK3CA / bon1 transgenic materials 15 days after treatment with 100mM mannitol. Scale bar, 0.5cm. (b) Phenotype of Col-0, bon1 and CPK3pro::CPK3CA / bon1 transgenic materials after 5 weeks of growth in soil. Scale bar, 2cm. (ce) Relative expression of PR1 (c) and PR2 (d) and the content of SA (e) in vivo of Col-0, bon1 and CPK3pro::CPK3CA / bon1 transgenic materials after 5 days of treatment with 100mM mannitol (unit: ng / g fresh weight). Values ​​are standard deviation ± SD, n = 3.

[0050] Figure 6 , CPK3 and SNC1TIR (a) Expression of SNC1 in leaves of eds1 tobacco mutants TIR -Myc, CPK3-YFP and YFP-YFP, co-immunoprecipitation with GFP magnetic beads, detection with Myc antibody, verifying the interaction between CPK3 and SNC1 TIR Interaction. *, CPK3-YFP; **, YFP-YFP; ***, SNC1 TIR (b) Luciferase complementation assay was used to detect the expression of CPK3 and SNC1 in leaves of eds1 tobacco mutants. TIR interaction.

[0051] Figure 7 、CPK3 phosphorylates SNC1 TIR (a) 32 In vitro phosphorylation assay using P-labeled ATP to analyze the effect of CPK3 on SNC1 TIR and SNC1 TIRAA(S18A / S26A) Phosphorylation modification of proteins. The upper picture shows autoradiography, and the lower picture shows Coomassie Brilliant Blue staining showing recombinant proteins GST-CPK3, GST-SNC1 TIR ,GST-SNC1 TIR-AA(S18A / S26A) (b) In vitro phosphorylation experiment using a specific phosphorylation antibody targeting serine 26 to verify that CPK3 phosphorylates SNC1 TIR Serine 26 of the mitochondria. λ-proteinphosphatase (λ-PPase) treatment was used as a negative control.

[0052] Figure 8 , osmotic activation of CPK3 phosphorylates SNC1 TIR Anti-thiophosphate ester antibody was used to detect phosphorylated SNC1 TIR Anti-SNC1 antibody was used to detect background SNC1 TIR .

[0053] Fig. 9 , CPK3 phosphorylates a conserved site in the TIR domain. (a) Sequence enrichment of 141 Arabidopsis proteins with TIR domains verified that the S26 site is highly conserved (above). Comparison of TIR proteins from various species and forms verified that TIR proteins are highly conserved in different species. (bd) CPK3 phosphorylates L6 TIR 、L6 TIRS66A 、RBA1、hSARM1 TIR and hSARM1 TIRS567A Phosphorylation modification of proteins. (b and d) Autoradiography (upper panel) and Coomassie Brilliant Blue staining showed that the recombinant proteins GST-CPK3 and His-L6TIR 、His-L6 TIR-S66A 、His-hSARM1 TIR and His-hSARM1 TIR-S567A (c) Anti-thiophosphate ester antibody was used to detect phosphorylated RBA1. Anti-GST was used to detect GST-CPK3. Anti-Flag was used to detect RBA1-Flag.

[0054] Fig.10 、CPK3 phosphorylates RPS4 TIR Autoradiography (upper panel) and Coomassie blue staining (lower panel) show phosphorylated and background recombinant proteins GST-CPK3 and GST-RPS4 TIR and GST-RPS4 TIRS33A .

[0055] Fig.11 、CPK4 / 5 / 6 / 11 phosphorylates SNC1 TIR And regulate plant growth. (a) CPK5 on SNC1 TIR and SNC1 TIRS18A / S26A(AA) Phosphorylation modification of proteins. Autoradiography (above) and Coomassie Brilliant Blue staining showed that the recombinant proteins GST-CPK5 and GST-SNC1 TIR and GST-SNC1TIR S18AS26A(AA) (b) CPK4 / 6 / 11 phosphorylates SNC1 TIR Autoradiography shows phosphorylated CPK4 / 6 / 11 and SNC1 TIR (c) Rosette leaf growth phenotypes of Col-0 and cpk4 / 5 / 6 / 11 quadruple mutants grown under 100 mM mannitol for 15 days. Scale bar, 0.5 cm.

[0056] Fig.12 The phosphorylation of serine sites in the TIR domain affects the function of the glutamate site at the structural level. TIR (PDB:5TEC) and hSARM1 TIR (PDB: 8D0J) schematic diagram. The conserved serine site and the catalytic glutamate site are shown in the schematic diagram, and the distance in the figure shows the distance between the carboxyl group of the glutamate site and the hydroxyl group of the serine site.

[0057] Fig.13 , phosphorylated TIR domain affects dimer formation. (a and b) Yeast two-hybrid experiments verify that mutations that mimic sustained phosphorylation (Ser-to-Asp) affect L6 TIRThe formation of TIR-TIR dimerization of RBA1 and GAL4-BD and GAL4-AD were respectively connected to L6 TIR and RBA1 and mutant L6 TIR and RBA1. Yeast interaction was verified in the triple-deficient medium lacking Leu, Trp, and His (-LWH) and the quadruple-deficient medium lacking Leu, Trp, His, and Ade (-LWH-Ade). (c and d) Western blot analysis of yeast cells in which GAL4-BD and GAL4-AD were respectively ligated into L6 TIR and RBA1 and mutant L6 TIR The Myc antibody was used to detect the expression of GAL4-BD and the linked fragment, and the HA antibody was used to detect the expression of GAL4-AD and the linked fragment.

[0058] Fig.14 、RBA1 digests NAD + Produce Nam and v-cADPR. (af) Mass spectrometry data confirmed that RBA1 can degrade NAD + Produces Nam and 2'cADPR, and the enzyme activity depends on the conserved glutamate site. (a, c, and e) Mass spectrometry peaks. (b, d, f) Mass spectrometry peak area statistics. Values ​​represent mean ± SEM (n = 3).

[0059] Fig.15 , phosphorylated TIR domain inhibits its NAD + Enzyme activity and the production of Nam and 2'cADPR. (a, c and e) are mass spectrometry peaks. (b, d and f) are mass spectrometry peak area statistics. Values ​​represent mean ± SEM (n = 3). (g) Quantification of protein expression of RBA1-Flag, RBA1S19A-Flag and RBA1S19D-Flag expressed in insect cells in in vitro enzyme activity assay.

[0060] Fig.16 , phosphorylated TIR domain inhibits NAD + Activity-dependent accumulation of 2'cADPR. Phosphorylation of the TIR domain inhibits NAD in plants + Activity-dependent 2'cADPR production. (a) RBA1, RBA1S19A, RBA1S19D and RBA1E86A were injected into tobacco leaves to simulate the continuous phosphorylation of RBA1S19D, which could not produce NAD+ activity-dependent 2'cADPR. (b) Protein expression of RBA1-YFP, RBA1S19A-YFP, RBA1S19D-YFP and RBA1E86A-YFP injected into tobacco leaves.

[0061] Fig.17、CPK3 CA Co-expression with TIR suppresses the hypersensitive phenotype of TIR-induced localized death in tobacco leaves. CA Co-expression with TIR inhibited TIR-induced localized tobacco leaf death, with TNL-TIRs alone injected as a control. (a) Different degrees of tobacco leaf death induced by TNL-TIRs. (b) SNC1 TIR 、L6 TIR Co-expression of CPK3 with RBA1 CA or expressed alone in tobacco leaves.

[0062] Fig.18 , Western blotting analysis to identify TNL-TIRs and CPK3 CA Expression of TNL-TIRs and CPK3 by Western blotting CA Expression in tobacco leaves. Flag antibody was used to detect CPK3 CA -cLUC-Flag protein expression. GFP antibody was used to detect SNC1 TIR -YFP and L6 TIR -YFP. HA antibody was used to detect RBA1-HA.

[0063] Fig.19 , simulated phosphorylated SNC1 TIR Inhibition of SNC1 TIR Induced tobacco leaf death. Phosphorylated SNC1 TIR Inhibition of SNC1 TIR Induced tobacco leaf death. (ac) Injection of SNC1 into tobacco leaves TIR -YFP, SNC1 TIRS18A -YFP, SNC1 TIRS18D -YFP, SNC1 TIRS26A -YFP, SNC1 TIRS26D -YFP, SNC1 TIRS18AS26A -YFP, SNC1 TIRS18D / S26D -YFP and SNC1 TIRE84A -YFP, observed in SNC1 TIR and mutation-induced tobacco leaf death.

[0064] Fig. 20 , Western blotting assay to identify SNC1 TIR (ad) Western blot analysis of wild-type SNC1 based on GFP antibody detection TIR The expression of the mutant in tobacco leaves. RuBisCO stained with Lichun Red was used as a control for the total protein loading.

[0065] Fig.21 Phosphorylated plant TNL-TIRs inhibited tobacco leaf death induced by TNL-TIRs. TIR and RBA1 inhibit TIR-induced tobacco leaf death. (a) L6 was expressed in tobacco leaves by injection TIR and mutation: L6 TIR -YFP, L6 TIRS66A -YFP, L6 TIRS66D -YFP and L6 TIRE135A (b) Wild-type RBA1-HA, RBA1S19A-HA, which mimics a constitutively inactive form of phosphorylation, RBA1S19D-HA, which mimics a constitutively phosphorylated form, and RBA1E86A-HA, which is a glutamate-inactive mutant, were expressed in tobacco leaves.

[0066] Fig. 22 , Western blot analysis to identify L6 TIR Protein expression of RBA1 and its mutants. Western immunoblot analysis of L6 TIR The protein expression of RBA1 and various mutant forms was detected by using RuBisCO stained with Lichun Red as a control. (a) GFP antibody was used to detect L6 TIR -YFP and its mutations. (b) HA antibody was used to detect RBA1 and its mutations.

[0067] Fig.23 Phosphorylation of TNL-TIRs inhibits tobacco leaf death induced by mammalian and bacterial TNL-TIRs. Phosphorylation of TNL-TIRs inhibits tobacco leaf death induced by mammalian and bacterial TNL-TIRs. (a) hSARM1 is expressed in tobacco leaves. tSAM-TIR -YFP, hSARM1 tSAM-TIRS567A -YFP, hSARM1 tSAM-TIRS567D -YFP and hSARM1 tSAM-TIRE642A -YFP. (b) Ab expression in tobacco leaves TIR -YFP, Ab TIRS140A -YFP, Ab TIRS140D -YFP and Ab TIRE208A -YFP.

[0068] Fig.24 , Western blot analysis of hSARM1 TIR Protein expression of hSARM1 mutants and mutants. Identification of hSARM1 by western blotting with GFP antibody TIR and hSARM1 TIR Expression of mutant proteins, and RuBisCO stained with Lichun Red as a control.

[0069] Fig.25, TNL-TIR phosphorylation modification is involved in plant growth maintenance. (a) Col-0, OE-SNC1-4Myc / Col-0 and OE-SNC1 S18D / S26D Phenotypes of transgenic materials of Col-0, OE-SNC1-4Myc / Col-0 and OE-SNC1 after 5 weeks of growth in soil. Scale bar, 2 cm. (b) Phenotypes of Col-0, OE-SNC1-4Myc / Col-0 and OE-SNC1 S18D / S26D Rosette leaf growth phenotype of transgenic materials of Col-0, OE-SNC1-4Myc / Col-0 grown under 100 mM mannitol treatment for 15 days. Scale bar, 0.5 cm. (ce) Col-0, OE-SNC1-4Myc / Col-0 and OE-SNC1 S18D / S26D Relative expression of PR1 (c) and PR2 (d) and SA (e) content in vivo in transgenic materials of -4Myc / Col-0 treated with 100 mM mannitol for 5 days. Values ​​are standard deviation ± SD. (c) and (d), n = 3.

[0070] Fig.26 , phosphorylated TNL-TIR partially inhibits plant immunity. (a) Pst DC3000 infects Arabidopsis Col-0, OE-SNC1-4Myc / Col-0 and OE-SNC1 S18D / S26D(DD) -4Myc / Col-0 leaf phenotype three days later. (b) Pst DC3000 infected Arabidopsis Col-0, OE-SNC1-4Myc / Col-0 and OE-SNC1 S18D / S26D(DD) -4Myc / Col-0 colony counts in leaves three days after infection. (c) Pst DC3000 infected Arabidopsis Col-0, OE-SNC1-4Myc / Col-0 and OE-SNC1 S18D / S26D(DD) -4Myc / Col-0 SA content in leaves three days later. (b) Values ​​represent standard deviation ± SD (n = 4). (c) Values ​​represent mean ± SEM (n = 3).

[0071] Fig. 27 , Schematic diagram of the model by which CPKs phosphorylate TIRs to inhibit their activity. DETAILED DESCRIPTION

[0072] After in-depth research, the inventors revealed for the first time that CPKs can phosphorylate TIR-NLRs, thereby inhibiting the increase of PR1 / PR2 induced by osmotic conditions in plants and the accumulation of SA content, giving plants the ability to regulate immunity and promoting plant growth under osmotic stress conditions.

[0073] When plants face a variety of stress stimuli, including biotic stress and abiotic stress, they will cause changes in calcium signals in the body. 2+Signal-related changes can affect plant growth and response to stress. The calcium signal decoding protein (calcium-dependent kinase) CPKs family in plants is an important element in plant response to biotic and abiotic stresses and in regulating plant growth. However, the downstream mechanism of action of CPKs of calcium signal sensor proteins is still poorly understood in this field. The new findings of the present invention provide a new way to further analyze the plant phosphorylation regulation mechanism and cultivate new varieties of plants with good stress resistance and both yield and growth.

[0074] As used herein, the "dicotyledonous plants" include (but are not limited to): flax plants, cruciferous plants, such as (but not limited to): cotton, hemp, soybean, peanut, sunflower, sweet potato, potato, pear, apple, tangerine, tobacco, mint, camphor, and nanmu.

[0075] The "flax plants" include (but are not limited to): flax.

[0076] The "cruciferous plants" include (but are not limited to): Arabidopsis, Chinese cabbage, kale, mustard, radish, and rapeseed.

[0077] It should be understood that, under the guidance of the technical solution of the present invention, those skilled in the art can easily think of changing the types of various plants to achieve the same or similar technical effects, and these changes are also included in the present invention.

[0078] CPKs-TIR-NLRs signaling pathway

[0079] As used in the present invention, the "(signal) pathway" refers to a signal system formed by a series of proteins or genes that restrict or interact with each other, which generally leads to the occurrence of some cellular events.

[0080] The CPKs-TIR-NLRs signaling pathway includes (but is not limited to): CPKs gene (and / or its encoded protein), TIR gene (and / or its encoded protein), SNC1 gene (and / or its encoded protein), TLRs gene (and / or its encoded protein), NLRs gene (and / or its encoded protein).

[0081] The CPKs-TIR signaling pathway includes (but is not limited to): CPKs gene (and / or its encoded protein), TIR gene (and / or its encoded protein).

[0082] As used in the present invention, the term "CPKs-TIR-NLRs signaling pathway" and "CPKs / TIR / NLRs signaling pathway" can be used interchangeably. The term "CPKs-TIR signaling pathway" and "CPKs / TIR signaling pathway" can be used interchangeably.

[0083] As used in the present invention, "signaling pathway", "(signal) module", "regulatory module" or "regulatory module" can be used interchangeably.

[0084] As used in the present invention, the "TIR" is a domain that is highly conserved and has relatively diverse functions. At the same time, it can be connected in series with proteins in other domains to sense and conduct signals. According to the type classification, TIR domains can be divided into three categories: the first category is composed of only one or several TIR domains, which exist in prokaryotes, oomycetes, plants and animals. This type of type contains only TIR domains (TIR-only) or other domains with very short tandem sequences, represented by vertebrate TIR proteins Myd88 and Mal, and RBA1 encoded by the Arabidopsis genome. The second category is that the TIR domain is connected to the STINGs domain, LRR domain, Ig domain or ank domain. This type of TIR domain is represented by animal TLR proteins, and oomycete cells also contain such proteins. The third type of TIR protein refers to a TIR domain that contains an NBD domain and an LRR domain or a WD40 domain, or a TPR domain. This type of protein is mainly represented by TIR-NBARC-LRR or TNLs in plants, which directly recognize effector proteins to activate immune signals and complete the plant's resistance to pathogens. The N-terminal domain of the classic TNLs protein mainly plays a signal transduction function, and its functionality depends on the oligomerization of the TIR domain. There is also a special NLRs protein in Arabidopsis, which functions in pairs, namely Pair-NLRs, including RPS4 / RRS1 and CHS3 / CSA1.

[0085] As used herein, the "TIR (domain)" includes TIR from Arabidopsis thaliana (At), genes or polypeptides that are homologous to genes or polypeptides from Arabidopsis thaliana, contain substantially the same domain, and have substantially the same function.

[0086] As used herein, the amino acid sequence of the "TIR (domain)" protein is, for example, as shown at positions 19-182 in the SNC1 protein UniProtKB / Swiss-Prot:O23530.5.

[0087] As used herein, the "CPKs gene" or "CPKs polypeptide" includes CPKs genes or polypeptides from Arabidopsis thaliana (At), genes or polypeptides that are homologous to genes or polypeptides from Arabidopsis thaliana, contain substantially the same structural domains, and have substantially the same functions.

[0088] In the present invention, the CPKs polypeptide, as one of the calcium signal decoding proteins in plants, has a total of 34 family members in Arabidopsis, including CPK3, CPK4, CPK5, CPK6, CPK11, etc.

[0089] The amino acid sequence of CPK3 is shown in GenBank: OAP00335.1, for example.

[0090] The amino acid sequence of CPK4 is shown in GenBank: OAP08681.1, for example.

[0091] The amino acid sequence of CPK5 is shown in, for example, GenBank: OAP00897.1.

[0092] The amino acid sequence of CPK6 is shown in GenBank: OAP08681.1, for example.

[0093] The amino acid sequence of CPK11 is shown in GenBank: OAP12436.1, for example.

[0094] Those skilled in the art know that, using the NCBI accession number, the nucleotide sequence of the desired gene and / or the amino acid sequence of the protein can be downloaded from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ).

[0095] In the present invention, the signaling pathway molecules (polypeptides) described herein also include their fragments, derivatives and analogs. As used herein, the terms "fragment", "derivative" and "analog" refer to protein fragments that substantially maintain the same biological function or activity of the polypeptide, which may be (i) a protein in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a protein having a substitution group in one or more amino acid residues, or (iii) a protein formed by the fusion of an additional amino acid sequence to this protein sequence, or (iv) a protein in which one or more amino acid residues (or domains) are removed (or truncated), for example, the self-inhibitory region and CaM-LD region of CPKs are removed, and only the variable region and kinase domain of the N-terminus are retained. According to the definition herein, these fragments, derivatives and analogs belong to the scope known to those skilled in the art. The biologically active fragments of the CPKs polypeptides described can be applied to the present invention.

[0096] In the present invention, the term "CPK3 polypeptide" or "CPK3 protein" refers to the protein shown in SEQ ID NO: 1 having CPK3 polypeptide activity. The term also includes variant forms of SEQ ID NO: 1 that have the same function as the CPKs polypeptide protein. These variant forms include (but are not limited to): deletion, insertion and / or substitution of several (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10, and even more preferably 1-8, 1-5) amino acids, and addition or deletion of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, the addition or deletion of one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein.

[0097] As used herein, the term "variant" or "mutant" or "mutant protein" or "variant form" or "Mutant" or "Mt" refers to a peptide or polypeptide whose amino acid sequence is changed by insertion, deletion or substitution of one or more amino acids compared to a reference sequence (wild-type sequence) but retains at least one biological activity; at the nucleic acid level, the corresponding coding site is changed compared to the corresponding reference sequence. In the present invention, "mutation" generally refers to a mutation that is closely related to the occurrence of a disease. The "inserted or substituted amino acid" can be a natural amino acid or a non-natural amino acid.

[0098] As used herein, "mutant CPK3" or "CPK3 mutant" or "CPK3 variant" means: a person skilled in the art, as needed, performs operations such as substitution or deletion or addition or mutation of one or more amino acids on the basis of SEQ ID NO: 1, and makes the obtained CPK3 protein variant have the same biological function as the CPK3 protein shown in SEQ ID NO: 1. The CPK3 protein variant may be a derivative protein having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more with SEQ ID NO: 1 and having the function of the protein shown in SEQ ID NO: 1. The "variant" includes truncations, such as but not limited to: a derivative protein having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more with SEQ ID NO: 2 and having the function of the protein shown in SEQ ID NO: 2.

[0099] The amino acid sequence of the CPK3 protein encoded by the CPK3 gene is shown in SEQ ID NO: 1, and the amino acid sequence of the truncated CPK3 protein encoded by the truncated CPK3 gene is shown in SEQ ID NO: 2.

[0100] As described in the present invention, the nucleotide sequence of the CPKs gene can be codon optimized according to actual conditions. The sequence of the CPKs gene of the present invention can be a nucleotide sequence after various optimizations. As long as it encodes the above-mentioned CPKs protein, or its protein variant, it belongs to the protection scope of the present invention. In some specific embodiments, the nucleotide sequence of the CPK3 gene is as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or has a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more thereto.

[0101] It should be understood that although the CPKs gene of the present invention is preferably obtained from a cruciferous plant, especially Arabidopsis, other genes obtained from other plants that are highly homologous to the Arabidopsis CPKs gene (e.g., having more than 80%, such as 85%, 90%, 95%, or even 98% sequence identity) or genes with degeneracy with the gene are also within the scope of the present invention. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0102] The vector containing the coding sequence, and the host cell produced by genetic engineering using the vector or the polypeptide coding sequence are also included in the present invention. Methods well known to those skilled in the art can be used to construct a suitable expression vector.

[0103] The host cell is usually a plant cell. Plant transformation can generally be carried out using methods such as Agrobacterium transformation or gene gun transformation, preferably Agrobacterium method. Transformed plant cells, tissues or organs can be regenerated into plants using conventional methods to obtain plants with altered traits relative to the wild type.

[0104] As used herein, the plant includes but is not limited to plants selected from the following groups: plants expressing CPKs genes or proteins; plants containing TIR domains; plants expressing SNC1 genes or proteins; plants expressing TNLs genes or proteins; plants expressing NLRs genes or proteins; dicotyledonous plants; and cruciferous plants.

[0105] When applied to regulating plant immunity and coping with osmotic stress-related traits, the "plant" includes plants expressing CPKs genes or proteins; plants containing TIR domains; plants expressing SNC1 genes or proteins; plants expressing TNLs genes or proteins or plants expressing NLRs genes or proteins. According to the knowledge in the art, plants with CPKs, TIR domains, SNC1, TNLs or NLRs have the mechanism of action claimed by the present invention, and can achieve the technical effects claimed by the present invention.

[0106] As used in the present invention, the "SNC1" is a TNL-type R protein, and the TNL-type R protein belongs to a class of NLR proteins. Therefore, SNC1 is also an NLR protein. The nucleotide sequence of the SNC1 is shown, for example, in Gene ID: 827397; its protein amino acid sequence is shown, for example, in UniProtKB / Swiss-Prot: O23530.5. The SNC1 has three domains, namely, a TIR domain, a NB-LRR domain and a LRR domain.

[0107] When used as a target for artificial regulation or when a screening system is artificially established, the above proteins or coding genes can be naturally occurring, such as being purified and isolated from mammals or plants; or they can be recombinantly prepared, such as recombinant proteins can be produced according to conventional gene recombination techniques. In addition, any variation that does not affect the biological activity of these proteins is available, such as derivatives or variants whose functions have not been changed.

[0108] Application of CPKs-TIR-NLRs signaling pathway

[0109] During the detailed analysis of the CPKs gene, the inventors found that the gene played an important role in plant immunity and response to osmotic stress.

[0110] In a specific embodiment of the present invention, using genetics, cell biology and biochemistry, the inventors found that calcium signal-activated CPKs can phosphorylate a highly conserved serine site (S26) of TIR-NLRs, and the phosphorylated form of TIR can inhibit its degradation of NAD + The enzyme activity of CPK3 can regulate the growth of plants in the immune process and stress response process. The constitutively activated form of CPK3 can restore the growth defect phenotype of plants under stress conditions caused by the activation of TIR-NLRs. The mutant form TIRSD that mimics the constitutively phosphorylated form can inhibit the TIR domain from activating NAD. +The degradation of 2'cADPR and the production of products such as 2'cADPR are inhibited, resulting in the loss of its function of inducing cell death phenotype. Osmotic stress is a typical early signal of abiotic stress, such as drought and salt stress, which can cause osmotic stress. Under long-term osmotic stress treatment, the expression of immune downstream marker genes PR1 / PR2 can be induced, and the SA level in the plant can also be increased, inhibiting the growth of plants under stress conditions. The CPK3-TIRs phosphorylation regulatory module can inhibit the increase of PR1 / PR2 induced by osmotic conditions in plants and the accumulation of SA content, and promote the growth of plants under osmotic stress conditions. Overexpression of TNLs can lead to plant autoimmune growth defects and growth defects in osmotic stress treatment, while inducing a continuous increase of PR1 / PR2 and accumulation of SA content in plants. TNLSD, which simulates continuous phosphorylation, can restore the defects of plant autoimmune phenotypes, while reducing the increase of PR1 / PR2 and SA accumulation induced by osmotic stress, and at the same time give plants a certain disease resistance, balancing the growth and resistance of plants under stress conditions.

[0111] Based on the above new findings of the inventors, the use of the CPKs-TIR-NLRs signaling pathway, its pathway molecules or their regulators is provided for: regulating the immune capacity of plants, including: enhancing (including maintaining) the ability of plants to respond to stress or tolerate stress, inhibiting (including maintaining) plant cell death, and promoting (including maintaining) plant growth.

[0112] In one or more embodiments, the regulator includes an up-regulator. The up-regulator includes: an agent that increases the phosphorylation level of TIR, an agent that promotes CPKs-TIR interaction and thus inhibits TIR; preferably, the agent that increases the phosphorylation level of TIR includes: an agent that increases the phosphorylation level of Ser at position 26 and / or position 18 of TIR, and more preferably includes an agent that mutates Ser to Asp (such as a point mutation agent, more specifically a point mutation construct / carrier material).

[0113] In one or more embodiments, the up-regulator includes: a CPKs up-regulator; more preferably includes: an exogenous CPKs encoding gene or an expression construct or vector containing the encoding gene, calcium ions (including calcium salts that can produce calcium ions), or an agent for performing a gain-of-function point mutation on CPKs.

[0114] The ability to regulate plant immunity described in the present invention includes: regulating plant disease resistance.

[0115] The disease resistance described in the present invention refers to the resistance of plants to plant diseases compared to the corresponding wild-type control. Plant diseases are usually caused by pathogens, including insects, fungi, bacteria, viruses, nematodes, viroids, mycoplasmas, etc. In one or more embodiments, the disease resistance includes: the ability to resist pathogens. The pathogens can be plant pathogens. The pathogens can be pathogens that can activate NLR protein signals containing TIR domains at the N-terminus.

[0116] "Plant pathogens" refers to any pathogen that causes a disease state in plants, including viruses, fungi, bacteria, nematodes and other microorganisms. Resistance or "disease resistance" refers to a reduction or attenuation of disease symptoms in plants after infection by pathogens. Symptoms can be diverse, but preferably include those that directly or indirectly adversely affect plant quality, yield, suitability as feed or food, or cause difficulties in sowing, planting, harvesting or processing of crops. Disease symptoms caused by pathogens include the size and / or frequency of necrotic or chlorotic lesions, brown lesions (which often have a light brown to slightly whitish center with a dark, slightly purplish to black outer edge, gradually surrounding the lesion) on plant tissues; the size and / or frequency of canker, black spot (also known as dark leaf spot), leaf downy mildew, etc.

[0117] The plant cell death described in the present invention includes plant cell death under stress conditions and plant cell death under non-stress conditions (such as autoimmune deficiency). The plant cell death includes but is not limited to plant cell apoptosis, necrosis (such as programmed necrosis), necroptosis, pyroptosis, cessation of growth, and death.

[0118] Plant growth described in the present invention includes plant growth under stress conditions, and also includes plant growth under non-stress conditions (such as caused by autoimmune deficiency). The "stress" includes: osmotic stress (high osmotic stress); preferably, the osmotic stress includes abiotic stress; preferably, the abiotic stress includes: salt (high salt) stress, cold stress, drought stress. The method according to the present invention can be beneficially combined with other methods, including causing pathogen resistance (such as insects, fungi, bacteria, nematodes, etc.), stress resistance or other improvements in plant characteristics (such as promoting plant growth).

[0119] The present invention also provides a method for screening substances (potential substances) that regulate plant disease resistance (immunity) traits, comprising:

[0120] (1) adding the candidate substance to a system expressing the CPKs-TIR-NLRs signaling pathway or its pathway molecules;

[0121] (2) detecting the system and observing the phosphorylation level of TIR therein; if the phosphorylation level is increased, it indicates that the candidate substance is a substance that can be used to regulate plant immunity.

[0122] In one or more embodiments, increasing the phosphorylation level of TIR comprises: increasing the phosphorylation level of Ser at position 26 and / or Ser at position 18 of TIR.

[0123] In one or more embodiments, the CPKs include CPK3, CPK4, CPK5, CPK6 and / or CPK11;

[0124] In one or more embodiments, the TIR comprises: a SNC1 protein, a TNLs protein or a NLRs protein containing a TIR domain.

[0125] In one or more embodiments, (2) includes: observing the effect of the candidate substance on the interaction between CPKs and TIR; if it promotes the interaction between the two and promotes CPKs to phosphorylate TIR, it indicates that the candidate substance is a substance that can be used to regulate plant immunity.

[0126] In one or more embodiments, the CPKs phosphorylating TIR includes: phosphorylating Ser at position 26 and / or position 18 of TIR.

[0127] Methods for screening substances that act on proteins or genes as targets are well known to those skilled in the art, and these methods can be used in the present invention. The candidate substances can be selected from: peptides, polymeric peptides, peptidomimetics, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, organic small molecules, inorganic small molecules and nucleic acid sequences, etc. Depending on the type of substance to be screened, those skilled in the art know how to select an appropriate screening method. After large-scale screening, a class of potential substances that specifically bind to CPKs and have a regulatory effect can be obtained.

[0128] The TIR domain and its S26 site are very conserved, which shows that their working mechanism is highly conserved. The inventors found that this site is not only highly conserved in 141 proteins with TIR domains in Arabidopsis, but also in other plant species (such as flax), bacteria (such as Ab protein ThsB with TIR domain) and mammals (such as hSARM1 TIR Based on the new discovery of the inventors, the TIR domain and its S26 site can be designed as bait proteins, and after the plants are subjected to osmotic stress, the components specific to the immune signaling pathway can be screened.

[0129] Therefore, the present invention provides a method for identifying plant immunity, comprising: analyzing the accumulation level of phosphorylated proteins of the plant's TIR domain and the protein phosphorylation level;

[0130] If the accumulation level of phosphorylated proteins in the TIR domain and / or the protein phosphorylation level is normal (or high), the plant immunity is normal (or low); if the accumulation level of phosphorylated proteins in the TIR domain and / or the protein phosphorylation level is abnormal (including disorder or down-regulation), the plant immunity is abnormal (or high); preferably, analyzing the protein phosphorylation level includes: analyzing the phosphorylation level of Ser at position 26 and / or Ser at position 18 of TIR;

[0131] Wherein, the regulating plant immunity includes: enhancing (including maintaining) the ability to respond to osmotic stress or the ability to tolerate stress, inhibiting plant cell death, and promoting (including maintaining) plant growth.

[0132] As used in this article, the normal and abnormal of the traits are relative. If the accumulation level of the TIR domain protein reaches the normal level, the TIR domain protein can be normally phosphorylated or the phosphorylation level of Ser at position 26 and / or Ser 18 of TIR reaches the normal level, then the trait is normal; otherwise, the trait is abnormal.

[0133] As described in this article, phosphorylation plays an important regulatory role in the normal function of proteins. This process is catalyzed by protein kinases (Kinase) to transfer the γ-position phosphate group of ATP or GTP to amino acid residues of substrate proteins, such as serine (Serine, Ser, S), threonine (Threonine, Thr, T) and tyrosine (Tyrosine, Tyr, Y), and the reverse process is the removal of the corresponding phosphate group by protein phosphatases. As described in this article, CPKs phosphorylate TIR domains, and increasing their phosphorylation levels is necessary for controlling plant immunity, indicating that the increase in the phosphorylation level of TIR domains is an important marker for regulating plant immune signaling pathways.

[0134] The present invention provides a method for regulating plant immunity, comprising: (1) increasing the gene or protein expression level of key kinases (such as CPKs) in the immune signaling pathway; and (2) increasing the phosphorylation level of important proteins (such as TIR domains) downstream of the key kinases.

[0135] Plant improvement applications

[0136] Based on the new discovery of the present inventors, a method for regulating plant immunity is provided, comprising: regulating (including upregulating) the CPKs-TIR-NLRs signaling pathway or its pathway molecules in the plant.

[0137] In one or more embodiments, plant immunity is regulated by upregulating CPKs-TIR-NLRs or pathway molecules thereof (promoting / activating the interaction of pathway molecules).

[0138] In one or more embodiments, regulating the plant's immune capacity includes: enhancing (including maintaining) the plant's ability to respond to stress or its tolerance to stress, inhibiting plant cell death, and promoting (including maintaining) plant growth.

[0139] In one or more embodiments, the upregulation of the CPKs-TIR-NLRs signaling pathway or its pathway molecules includes: increasing the phosphorylation level of TIR, promoting CPKs-TIR interaction to inhibit TIR, and regulating the immune capacity of the plant; preferably, increasing the phosphorylation level of TIR includes: increasing the phosphorylation level of Ser at position 26 and / or position 18 of TIR; preferably, the phosphorylation level is increased by mutating Ser to Asp.

[0140] In one or more embodiments, the upregulation includes: enhancing the ability of CPKs to phosphorylate TIR; transferring the coding gene of CPKs or an expression construct or vector containing the coding gene into the plant; activating CPKs with calcium ions (including calcium salts that can produce calcium ions); performing gain-of-function mutations on CPKs; promoting the expression of CPKs with an expression-enhancing promoter or a tissue-specific promoter; or promoting the expression of CPKs with an enhancer.

[0141] In one or more embodiments, the CPKs include CPK3, CPK4, CPK5, CPK6, and CPK11.

[0142] In one or more embodiments, the TIR comprises: a SNC1 protein, a TNLs protein or a NLRs protein containing a TIR domain.

[0143] It should be understood that after knowing the function of the signal pathway containing CPKs (preferably, also including its upstream genes and downstream genes), various methods well known to those skilled in the art can be used to regulate the expression or activity of the CPKs or regulate its upstream genes or downstream genes. For example, various methods well known to those skilled in the art can be used to recombinantly express or overexpress CPKs. These methods are all included in the present invention.

[0144] In the present invention, the reagents for up-regulating the CPKs protein or its coding gene, its upstream or downstream protein or its coding gene can be called up-regulators, which include promoters, agonists, and activators. The "up-regulation" and "promotion" include "up-regulation" and "promotion" of protein activity or "up-regulation" and "promotion" of protein expression, and they are statistically significant "up-regulation" and "promotion". Any substance that can increase the activity of CPKs or signaling pathway proteins containing them (including upstream and downstream proteins), increase the stability of CPKs or signaling pathway proteins containing them, up-regulate the expression of CPKs or signaling pathway genes containing them, increase the effective action time of CPKs or signaling pathway proteins containing them, and increase the phosphorylation / activation level of each protein can be used in the present invention as a substance useful for up-regulating CPKs or signaling pathways. They can be compounds, small chemical molecules, and biological molecules. The biological molecules can be at the nucleic acid level (including DNA, RNA) or at the protein level.

[0145] The main advantages of the present invention are:

[0146] The inventors have conducted in-depth research on the action mechanism of CPKs in the immune signaling pathway in plants and found that CPKs can phosphorylate TIR-NLRs, thereby inhibiting the increase of PR1 / PR2 induced under osmotic conditions in the plant body and the accumulation of SA content, giving plants the ability to regulate immunity and promoting plant growth under osmotic stress conditions. This has important application value for the transformation of plant cell signaling pathways and ultimately achieving plant resistance to osmotic stress and pathogen invasion.

[0147] When plants face a variety of stress stimuli, including biotic stress and abiotic stress, they will cause changes in calcium signals in the body. 2+ Signal-related changes can affect plant growth and response to stress. The calcium signal decoding protein (calcium-dependent kinase) CPKs family in plants is an important element in plant response to biotic and abiotic stresses and in regulating plant growth. However, the downstream mechanism of action of CPKs of calcium signal sensor proteins is still poorly understood in this field. The new findings of the present invention provide a new way to further analyze the plant phosphorylation regulation mechanism and cultivate new varieties of plants with good stress resistance and both yield and growth.

[0148] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd Edition, Science Press, or according to the conditions recommended by the manufacturer.

[0149] Materials and methods

[0150] 1. Experimental Materials

[0151] The mutant materials used in the present invention include Col-0, snc1-1 (Zhang et al., 2003), bon1-1, bon1-7 (Chen et al., 2020), AEQsig6 (Chen et al., 2020), and cpk4 / 5 / 6 / 11 quadruple mutants. The ios mutant was screened from a mutant library constructed by EMS mutagenesis with bon1-7 as the background. Specifically, it was screened based on the growth phenotype after growing on 100 mM mannitol for 15 days and in soil for 5 weeks. The tobacco materials used in the present invention are wild-type N. benthamiana and eds1-2 mutants (Gantner et al., 2019).

[0152] Construct the transgenic material of OE-SNC1-4Myc / Col-0, clone the genome sequence of AtSNC1 (Gene ID: 827397, including the entire coding region from the start codon ATG but not the stop codon) into pCambia1305-35S-4Myc to obtain the binary transformation vector of pCambia1305-35S-SNC1-4Myc, and obtain the positive clone by sequencing the bacterial solution and plasmid. Then infect the Col-0 Arabidopsis material by Agrobacterium-mediated floral dip method. The obtained transgenic material is screened on hygromycin, and the transgenic material is identified by PCR and western blot based on Myc antibody. The TIR domain of SNC1 protein is 10-235 in UniProtKB / Swiss-Prot:O23530.5 (the blue part of the amino acid sequence below):

[0153] TIR region of SNC1 amino acid sequence (SEQ ID NO:3, positions 10-235):

[0154] MMDTSKDDDMEIASSSGSRRYDVFPSFRGEDVRDSFLSHLLKELRGKAITFIDDEIERSRSIGPELLSAIKESRIAIVIFSKNYASSTWCLNELVEIHKCYTNLNQMVIPIFFHVDA SEVKKQTGEFGKVFEETCKAKSEDEKQSWKQALAAVAVMAGYDLRKWPSEAAMIEELAEDVLRKTMTPSDDFGDLVGIENHIEAIKSVLCLESKEARIMVGIWGQSGIGKSTIGRALY

[0155] Build OE-SNC1 S18D / S26D -4Myc / Col-0 (wherein, S18D is the mutation of the amino acid residue at position 18 of the protein encoded by SNC1 from A to D, and S26D is the mutation of the amino acid residue at position 26 of the protein encoded by SNC1 from A to D). The transgenic material is a vector of pHBT-SNC1 obtained by a Transfer-PCR amplification method using pCambia1305-35S-SNC1-4Myc plasmid as a template, and pHBT-SNC1 is obtained by a point mutation amplification method using pHBT-SNC1 as a template. S18D / S26D Then, pCambia1305-35S-SNC1 was obtained by restriction digestion and ligation. S18D / S26D -4Myc binary vector. Positive clones were obtained by bacterial culture and plasmid sequencing. Col-0 Arabidopsis thaliana material was then infected by Agrobacterium-mediated floral dip method. The obtained transgenic material was screened on hygromycin and identified by PCR and western blot based on Myc antibody.

[0156] Build OE-SNC1 G1343D / bon1-7 (where G1343D is the amino acid residue at position 1343 of the protein encoded by SNC1 mutated from G to D) and OE-SNC1 V518I / bon1-1 (wherein V518I is the mutation of the amino acid residue at position 518 of the protein encoded by SNC1 from V to I), and pHBT-SNC1 plasmid was used as a template to obtain pHBT-SNC1 by point mutation. G1343D and pHBT-SNC1 V518I The vector plasmid was then obtained by PCR amplification, restriction digestion and ligation to obtain pCambia1305-35S-SNC1 G1343D -4Myc and pCambia1305-35S-SNC1 V518I -4Myc binary transgenic vector. Positive clones were obtained by bacterial solution and plasmid sequencing. Then, mutant bon1-7 or bon1-1 Arabidopsis thaliana was infected by Agrobacterium-mediated floral dip method. The obtained transgenic materials were then screened on hygromycin and the transgenic expression was identified by Real-Time PCR.

[0157] The transgenic material of CPK3proCPK3-4Myc / Col-0 was constructed, and the genomic sequence of AtCPK3 (GeneID: 828465, including the promoter region of 2000bp and the entire coding region, excluding the stop codon) was cloned into the binary vector of pCambia1305-4Myc, and the positive clones were subjected to bacterial liquid and plasmid sequencing. Then, the Col-0 Arabidopsis material was infected by the Agrobacterium-mediated floral dip method. The obtained transgenic material was screened on hygromycin, and the transgenic material was identified by PCR and western blot based on Myc antibody.

[0158] Constructing CPK3proCPK3 CA -4Myc / bon1-1 transgenic material, the truncated CPK3 genomic sequence (encoding truncated AtCPK3 protein, including 2000bp promoter region, and CPK3CA sequence (truncated sequence of CPK3 coding region), excluding stop codon) was cloned into the binary vector of pCambia1305-4Myc, and the positive clones were sequenced by bacterial liquid and plasmid. Then, the mutant bon1-1 Arabidopsis material was infected by Agrobacterium-mediated floral dip method. The obtained transgenic material was screened on a culture medium containing hygromycin, and the transgenic material was identified by Real-Time PCR.

[0159] AtCPK3 protein sequence (SEQ ID NO: 1)

[0160] MGHRHSKSKSSDPPPSSSSSSSGNVVHHVKPAGERRGSSGSGTVGSSGSGTGGSRSTTSTQQNGRILGRPMEEVRRTYEFGRELGRGQFGVTYLVTHKETKQQVACKSIPTRRLVHKDDIEDVRREVQIMHHLSGHRNIVDLKGAYEDRHSVNLIMELCEGGELFDRIISKGLYSERAAADLCRQMVMVVHSCHSMGVMHRDLKPENFLFLSKDENSPLKATDFGLSVFFKPGDKFKDLVGSAYYVAPEVLKRNYGPEADIWSAGVILYILLSGVPPFWGENETGIFDAILQGQLDFSADPWPALSDGAKDLVRKMLKYDPKDRLTAAEVLNHPWIREDGEASDKPLDNAVLSRMKQFRAMNKLKKMALKVIAENLSEEEIIGLKEMFKSLDTDNNGIVTLEELRTGLPKLGSKISEAEIRQLMEAADMDGDGSIDYLEFISATMHMNRIEREDHLYTAFQFFDNDNSGYITMEELELAMKKYNMGDDKSIKEIIAEVDTDRDGKINYEEFVAMMKKGNPELVPNRRRM

[0161] Truncated AtCPK3 protein sequence (SEQ ID NO:2)

[0162] MGHRHSKSKSSDPPPSSSSSSSGNVVHHVKPAGERRGSSGSGTVGSSGSGTGGSRSTTSTQQNGRILGRPMEEVRRTYEFGRELGRGQFGVTYLVTHKETKQQVACKSIPTRRLVHKDDIEDVRREVQIMHHLSGHRNIVDLKGAYEDRHSVNLIMELCEGGELFDRIISKGLYSERAAADLCRQMVMVVHSCHSMGVMHRDLKPENFLFLSKDENSPLKATDFGLSVFFKPGDKFKDLVGSAYYVAPEVLKRNYGPEADIWSAGVILYILLSGVPPFWGENETGIFDAILQGQLDFSADPWPALSDGAKDLVRKMLKYDPKDRLTAAEVLNHPWIREDGE

[0163] 2. Plant growth conditions

[0164] Most of the growth conditions of Arabidopsis materials in the present invention are long-day (16 hours of light and 8 hours of darkness), the growth temperature is set at 22°C, and the relative humidity is 60-70%. This condition is suitable for the phenotypic observation of Arabidopsis after 5 weeks of growth in the soil. Arabidopsis seeds are disinfected with 5% sodium hypochlorite for 10 minutes before the experiment, and then rinsed 5 times with sterile water in a clean bench. The seeds are spotted on a medium of 1 / 2MS (Phytotech, M524), 1% sucrose, 1.2% agar, pH5.7. The culture dish is placed in a 4°C cold storage for 3 days. For transplanting and growing under mannitol conditions, the culture dish with seeds spotted and placed in a 4°C cold storage for 3 days is placed in a long-day incubator (Percival CU36L5, 16 hours of light, 8 hours of darkness, and a temperature of 22°C) and grown for 3 days before transplanting to a 1 / 2MS culture dish containing mannitol. This operation is suitable for the observation of growth phenotype under mannitol treatment, detection of PR1 / PR2 marker genes and determination of SA content. For the Arabidopsis materials used for the pst DC3000 inoculation experiment, Arabidopsis was grown in a short-day light box (10 hours of light and 14 hours of darkness), the growth temperature was set at 22°C, and the relative humidity was 60-70%.

[0165] The basic growth conditions of the tobacco material used in the present invention are set in an artificial climate chamber at a temperature of 22°C and a light condition of 12 hours of light and 12 hours of darkness. The tobacco is used for TNL-TIRs-induced tobacco leaf death phenotype observation, Co-IP experiment, Split-Luc experiment and mass spectrometry experiment after growing for about 5 weeks.

[0166] 3. Gene localization of ios mutants

[0167] Using bon1-7 as the background, EMS mutagenesis was carried out to construct a bon1-7-mutant mutant library. The mutant library of bon1-7-mutant was first screened under 100mM mannitol growth conditions (the screening criterion was to restore the sluggish phenotype of the cotyledons of the bon1 mutant under mannitol treatment conditions), and then the ios mutant was screened to restore the dwarf phenotype of the autoimmunity of the bon1 mutant after growing in the soil for 5 weeks. The ios mutant was backcrossed with bon1-7 to obtain the F1 material. The F1 material was grown under mannitol for 15 days and was found to be consistent with the ios mutant phenotype. At the same time, it was found to be the same as the ios mutant after growing in the soil for 5 weeks, so the gene mutation type of the ios mutant is a dominant mutation. The F1 material was multiplied to obtain the F2 population.

[0168] For the F2 population material, 50 strains were selected that had the same slow growth as the bon1-7 mutant under mannitol conditions, and the same growth phenotype as bon1-7 after growing in the soil for 5 weeks, to obtain a sequencing population. For the 50 selected F2 populations, leaves of the same mass were selected from each plant by weighing, and then the leaves of the same mass were mixed, and the leaf DNA was extracted by CTAB method, and the extracted DNA was sent to the gene department of Meiji Company for second-generation sequencing. The extracted DNA was sequenced on the Meiji sequencing platform to construct a sequencing library. The sequencing library was spliced ​​with a length of 400bp per segment, and the splicing system was Illumina HiSeq system (PE150). Gene markers were selected using the QTL-seq method. The position of the SNP on the chromosome was determined by a step-by-step sliding window method. The specific position of the SNP related to the phenotype was obtained by the calculation method of ΔSNP. The analysis results were verified. 50 F2 populations were selected for reproduction and the phenotype was reconfirmed to obtain the F3 population.

[0169] DNA was extracted from the F3 population, and PCR and sequencing were performed based on the obtained analysis results. For the candidate gene, the relevant transgenic vector was constructed, and the bon1 mutant was infected by the transgenic method of floral dipping. The relevant phenotypic analysis was performed on the obtained transgenic material to determine the accurate positioning.

[0170] 4. Agrobacterium-mediated tobacco transformation experiment

[0171] Mix the constructed binary vector with GV3101. 5 min on ice, 5 min in liquid nitrogen, 5 min at 37°C, and 5 min on ice again. Add 1 mL of LB without antibodies and culture in a 28°C incubator for 3 h. Spread the bacterial solution evenly on the double-antibody culture medium. Culture in a 28°C incubator for 3 days. Perform single colony identification, pick positive clones and culture them overnight in a 28°C shaker in a double-antibody liquid culture medium. Centrifuge and enrich the overnight cultured bacterial solution. Resuspend the enriched Agrobacterium with tobacco transformation liquid, which is 10 mM MgCl2, 10 mM MES, pH 5.6 and 150 μM acetosyringone. Calibrate the concentration at OD600nm. In order to prevent gene silencing, 35S:P19 with OD600nm=0.1 can also be added. Take tobacco grown for about 5 weeks for injection transformation experiments.

[0172] 5. GST-tagged prokaryotic protein purification

[0173] Mix the constructed GST tag vector (pGEX4T-1) and BL21 expression competent cells. Let stand on ice for 10 minutes, 42℃ for 1 minute, and on ice for 5 minutes. Add 1mL of LB without antibiotics and culture at 37℃ for 1 hour. Spread the bacterial solution evenly on the culture medium with antibiotics. Invert and culture overnight to identify positive clones. Pick a single colony and put it into 5mL of liquid culture medium with antibiotics and culture it at 37℃ overnight. Expand the 5mL bacterial solution from the overnight culture and culture it on a shaker at 37℃ for 3 hours. Add IPTG with a final concentration of 1mM and culture it at 18℃ for 14-16 hours. Enrich the cultured bacterial solution at 80000g in a 4℃ centrifuge. Resuspend it with Lysis buffer. Ultrasonicate it at 4℃. Centrifuge the broken bacterial solution at 20000g in a 4℃ centrifuge. Take the supernatant after centrifugation and incubate it with the pre-equilibrated GST-beads for 3 hours. Centrifuge at 4°C at 500 g to enrich beads. Remove supernatant. Wash 3 times with Lysis Buffer. Add appropriate amount of elution buffer to compete beads. Measure the concentration of each eluted protein and aliquot, store at -80°C.

[0174] 6. Based on 32 In vitro phosphorylation assay of P

[0175] Prepare kinase buffer stock solution (25mM Tris-HCl, pH7.5, 10mM MgCl2, 0.25mM DTT). Add the purified protein in advance to 20μL reaction system according to the appropriate ratio. 32 Add appropriate amount of 12.5 μM ATP. React at 30℃ for 3 hours. Add protein loading buffer. Boil at 98℃ for 5 minutes. Perform SDS-PAGE electrophoresis. Stain with Coomassie Brilliant Blue and take pictures. Dry the gel for 2 hours in a gel dryer. Press the dried gel under a phosphor screen overnight. Remove the phosphor screen and scan it in a scanner (radioautography).

[0176] 7. In vitro phosphorylation experiment based on PNBM alkylation

[0177] Prepare kinase buffer (25mM Tris-HCl, pH7.5, 10mM MgCl2, 0.25mM DTT). Mix the appropriate concentration of protein with kinase buffer. Add ATPγS to a final concentration of 0.5mM. React at 30℃ for 30min. Add PNBM to a final concentration of 2.5mM. React at 30℃ for 1 hour. Add protein loading buffer. 98℃, 5min. Perform western blot.

[0178] 8. Co-IP Experiment

[0179] Construct 35S:SNC1 TIR -Myc, 35S:CPK3-YFP and 35S:YFP-YFP binary expression vectors. Injection expression was performed in tobacco of about 5 weeks old through Agrobacterium-mediated transformation experiments. Three days after injection, the leaves were cut and snap-frozen in liquid nitrogen. The leaves were ground in IP beffer (20mM Tris–HCl, pH 7.5, 150mM NaCl, 5mM DTT, 0.5% NP40 and 1×proteaseinhibitor cocktail). Centrifuge at 4°C, 12000g for 40min. Take the supernatant and incubate with GFP-beads at 4°C for 3 hours. At the end of incubation, rinse with IP buffer 5 times. Rinse with PBS 3 times. Perform western blot experiments based on Myc antibodies and GFP antibodies.

[0180] 9. Split-Luc experiment

[0181] SNC1 TIR The coding regions of luciferase and CPK3 were connected into pCAMBIA1300-cLUC and pCAMBIA1300-nLUC, respectively. Through Agrobacterium-mediated tobacco transformation experiment, tobacco grown for about 5 weeks was injected for expression. Three days after injection, Luc substrate was incubated and Luc signal was observed under CCD.

[0182] 10. Immunoprecipitated kinase experiment

[0183] 7-day-old Arabidopsis seedlings were treated with 0.6 M mannitol for 30 min. The treated Arabidopsis were quickly frozen in liquid nitrogen. The samples were ground on ice and IP buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween-20, 1 mM NaF, 1 mM Na3VO4, 1 mM EDTA (pH 8.0) and InStab TMContinue grinding with Phosphatase Inhibitor Cocktail until liquid. Transfer to a centrifuge tube and centrifuge at 12000g at 4℃ for 40min. Combine the supernatant with the beads and incubate at 4℃ for 3 hours. After incubation, wash 3 times with IP buffer. Wash 3 times with Kinase buffer. Mix the beads with the prokaryotic substrate protein in kinase buffer. 32 P or in vitro phosphorylation experiment based on PNBM alkylation.

[0184] 11. In vitro NAD + Enzyme activity assay

[0185] Mix the purified protein and final concentration of 30μM NAD+ in enzyme activity reaction solution (92.4mM NaCl and 0.64XPBS). React at 25℃ for 2 hours. Add 1M HClO4. Place on ice for 10 minutes. Add 3M K2CO3 to neutralize the reaction. Place on ice for 10 minutes. Add 50% methanol. Perform LC-MS / MS experiment.

[0186] 12. Infection experiment of plant pathogenic bacteria Pseudomonas syringae pst DC3000

[0187] Streak the pst DC3000 bacterial solution stored at -80℃ onto LM medium containing rifampicin antibiotics. Pick a single colony and place it in liquid LM medium containing rifampicin antibiotics. Culture overnight at 28℃. Collect the bacterial solution and wash it twice with sterile water. Resuspend it with sterile water and adjust OD600nm=0.002. Inject the bacterial solution into Arabidopsis leaves. Put the Arabidopsis at room temperature for 2 hours and blow dry the surface liquid of the Arabidopsis after injection. Infect under high humidity conditions for 2 days. Take pictures to observe the phenotype, collect the injected Arabidopsis leaves, and place them in 75% ethanol for 30s. After drying with paper, the leaves are placed in sterile water for 2 times. Dry with paper and sample the injected leaves with a hole punch. Place the sample in a centrifuge tube with 200μL of sterile water added in advance. Shake and break it in a crusher at 40Hz. Add 800μL of sterile water again. After shaking and mixing, 5000g for 30s. Take the supernatant and dilute it in order. Take 10 μL of the diluted supernatant and evenly drop it on the LM medium containing rifampicin resistance. Incubate at 28°C for 3 days. Count the number of colonies under a microscope.

[0188] 13. Determination of SA content in plants

[0189] Weigh 0.05 g of plant material and freeze it in liquid nitrogen. Shake the sample with a grinder at an amplitude of 50 Hz. Add 400 μL of ethyl acetate to the tube containing the shattered sample. Vortex to mix and make sure the precipitate is completely mixed. Centrifuge at 4°C, 15,000 rpm for 5 minutes. Take the supernatant of the upper layer formed by the ethyl acetate and water layer. Add another 300 μL of ethyl acetate to the tube with the precipitate. Vortex to mix and make sure the precipitate is completely mixed. Centrifuge at 4°C, 15,000 rpm for 5 minutes. Take the supernatant of the upper layer formed by the ethyl acetate and water layer and mix it with the sample in step 6. Repeat steps 7-10. Blow liquid nitrogen. Add 100 μL of 30% methanol-water mixture to the dried sample to reconstitute the sample. Vortex the tube for 30 seconds, sonicate for 30 seconds, and vortex again for 30 seconds. Centrifuge at 4°C, 15,000 rpm for 10 minutes. Pipette 80 μL of supernatant, continue centrifugation, take out 70 μL and add it to the sample loading bottle for LC-MS / MS analysis.

[0190] 14. Purification of RBA1-Flag in insect expression cells

[0191] RBA1 (UniProtKB / Swiss-Prot: F4HT77.1, 1-191 amino acids) and the point sequence of RBA1 were constructed into pFastBac1 (Invitrogen). Take 0.5-1μL of plasmid and add 100μL DH 10Bac competent medium and mix well. Ice bath for 30min. Place at 42℃ for 45s. Ice bath for 2min. Add 1mL of antibiotic-free LB and incubate at 37℃ for 1 hour. Centrifuge at 3000rpm for 2min, discard the supernatant, and about 100μL remains. Puff evenly and spread on LB medium containing three antibiotics: Kan, Tet, and Gen. At the same time, IPTG and X-gal are added to the LB medium. Culture for 36-48 hours until white colonies are seen. Pick white colonies, identify positive clones by colony, and propagate by shaking. Extract Bacmid plasmid.

[0192] Place the extracted Bacmid plasmid on ice to melt, take 20-30μg Bacmid plasmid and 4μL cellfectin and dissolve them in 100μL Grace medium (this medium does not contain serum and has no resistance), and mix the two together. Incubate at room temperature for 30 minutes to fully mix the DNA and transfection reagent. Spread the suspended sf9 cells evenly in the cell culture dish, blow dry for 1 hour, and remove the excess liquid. Add 800μL Grace medium and mix with the Bacmid-Cellfectin mixture prepared in advance. After standing for 5 hours, remove the excess liquid and add the culture medium (ESF921). After 96 hours of culture, collect the P1 generation virus. Take the suspended sf9 cells and add them to the culture dish, and add the P1 generation virus at the same time. After 72 hours of culture, collect the P2 generation virus. Take the suspended sf9 cells and add them to the culture dish, and add the P2 generation virus at the same time. After 72 hours of culture, collect the P3 generation virus. Shake slightly and add the P3 generation virus to the cells in the subculture. Shake vigorously. Add the virus-cell liquid from small shakes to a large cell culture system. Collect the shaken cell liquid and centrifuge at 8000rpm, 4℃, 5min. Add cell lysis buffer (50mM Tris-HCl, pH, 7.5, 300mM NaCl, 10% glycerol, 5mM ATP, 1mM EDTA, 0.2% Chaps) to the collected bacterial mass. Add 1mg / mL lysozyme, 100mMPMSF, 2mM DTT to the above liquid, and set aside a small portion of the sample. Ultrasonic disruption. Centrifuge at 4℃, 20000g for 1h. Take the supernatant and add it to the filter membrane for filtration. Mix the filtered supernatant with the pre-equilibrated beads. Incubate at 4℃ for 2 hours. After the incubation, centrifuge at 4℃, 1000rpm for 10min. Remove the supernatant and wash 3 times with lysis buffer. Add elution buffer to elute the beads twice, eluting for 30min each time. After elution, centrifuge at 4°C, 1000 rpm for 10 min, collect the supernatant, and detect protein expression by western blot.

[0193] 15. TNL-TIRs-induced tobacco leaf death phenotype

[0194] Use Agrobacterium-mediated tobacco transformation to inject expression into tobacco leaves of about 5 weeks old. Two days after injection, use a hole puncher to punch holes in tobacco leaves and take samples, and store the samples at -80°C. Three days after injection expression, take leaves for observation. Place the leaves in a photo box and take pictures of the leaves with a camera under visible light. Quickly place the leaves under ultraviolet conditions and quickly take pictures with a camera in the photo box. For the tobacco samples in step 2, perform a western blot experiment to determine the expression level.

[0195] 16. Yeast two-hybrid experiment

[0196] RBA1 and L6 TIR Wild-type and mutant sequences were cloned into pGBKT7 and pGADT7. The stored Y2H Gold strain was streaked and activated on YPDA solid medium. Grow at 30°C for 2-3 days. Pick a single clone and inoculate it into 5mL liquid YPDA medium, and culture it at 30°C, 250rpm for 12 hours. Add part of the overnight culture to 50ml liquid YPDA medium and continue to culture at 30°C, 250rpm until OD600nm=0.6-0.8. Collect the culture and centrifuge at 2500rpm for 5min at room temperature. Remove the supernatant and wash twice with sterile water. Centrifuge at 2500rpm for 5min at room temperature for the last time and remove the supernatant. Resuspend the precipitate with 1X TE / LiAC (about 100μL yeast competent medium is required for each sample) and divide the plasmid into aliquots, mix them together according to 500ng of each plasmid, and add 10μL salmon sperm DNA (10mg / mL). Add 100μL TE / LiAC to each tube of mixed plasmid and resuspend competent cells. Add 600μL PEG / LiAC, pipette and mix thoroughly, and culture at 30℃, 250rpm for 30min. Add 70μL DMSO to each tube, mix well, and water bath at 42℃ for 15min. Centrifuge at 700g for 5min at room temperature and remove supernatant. Resuspend cells with 500μL TE, spread on a plate, and grow for 2-3 days. Pick a single colony and spot it on SD / -Leu, -Trp, SD / -Leu, -Trp, -His and SD / -Leu, -Trp, -His, -Ade culture media to observe colony growth.

[0197] 17. Preparation of phosphorylation antibody specific to S26 site

[0198] The specific phosphorylated antibody against the S26 site was produced by Wuhan ABclonal Technology. The C-DVFP(Sp)FRGED modified peptide was purified to a high purity by HPLC. And the modified peptide was used as an antigen to prepare a specific phosphorylated antibody against the S26 site. At the same time, the specific phosphorylated antibody against the S26 site was absorbed by the control peptide C-DVFPSFRGED (non-modified) to complete the purification.

[0199] Example

[0200] Example 1: Dominant negative mutation of SNC1 can inhibit NLR signaling to maintain plant growth

[0201] BON1 protein inhibits the NLRs signal of plants and is the main positive regulator involved in regulating plant response to osmotic stress and the negative regulator involved in regulating plant immunity. The bon1 mutant exhibits a typical phenotype of slow cotyledon growth under long-term osmotic stress. On the other hand, the continuously activated NLR signal in the bon1 mutant causes the plant to exhibit a dwarf phenotype of immune self-activation when grown in the soil. In order to explore how the BON1 protein affects the NLR signal and regulates plant growth, the bon1 mutant was induced with EMS and a mutant library was constructed. Under mannitol treatment (osmotic stress), the mutant library was transplanted and screened to obtain suppressors of the bon1 mutant. These suppressors can restore the phenotype of slow cotyledon growth of the bon1 mutant under osmotic treatment ( Figure 1 b and d), these mutants were named ios (insensitive toosmotic stress). At the same time, it was found that these ios mutants could restore the dwarf phenotype of bon1 mutants in soil due to immune self-activation ( Figure 1 c and e).

[0202] The ios mutants were backcrossed with the bon1 mutants, and the phenotype of the F1 was analyzed. The phenotype of the F1 was consistent with that of the ios mutants, so the phenotype of these ios mutants was caused by dominant mutations. Further, by isolating the phenotype of the F2 population, and performing positional cloning and BSA pooled second-generation sequencing analysis on the F2 population, the inventors located that the mutant genes of the six ios mutants were all allelic mutations of the TNL-type R protein SNC1 ( Figure 1 a). Therefore, these ios mutants were renumbered as ios22 snc1-16 、ios26 snc1-17 、ios102 snc1-18 、ios109 snc1-19 、ios112 snc1-20 and ios113 snc1-21 ( Figure 1 a). At the same time, the inventors constructed OE-SNC1 G1343D / bon1-7 and OE-SNC1 V518I / bon-1 complement transgenic materials, and found that they can simultaneously restore the cotyledon development retardation of the bon1 mutant under osmotic stress and the self-immune dwarf phenotype in the soil ( Figure 1 be).

[0203] In order to further confirm that SNC1 is involved in regulating plant growth under osmotic stress, the inventors conducted phenotypic analysis of the snc1-1 mutant material under osmotic stress. The snc1-1 mutant has an autoimmune dwarf developmental phenotype, and under osmotic stress treatment conditions, the inventors found that snc1-1 has a phenotype of delayed rosette leaf development consistent with the bon1 mutant ( Figure 3 ). Compared with the wild type, in the bon1 mutant, the typical marker genes PR1 and PR2 in the NLR signaling have a sustained high expression under osmotic stress treatment. Therefore, the inventors detected the expression of the PR1 gene in the suppressor and the transgenic complementation material under osmotic stress treatment. The inventors found that both ios22 and the transgenic complementation material can restore the sustained high expression of PR1 / PR2 in the bon1 mutant under osmotic stress treatment conditions ( Figure 2 a and b). At the same time, compared with the wild type, the SA content of the bon1 mutant continued to increase under osmotic stress treatment, while ios22 and transgenic complementation materials restored the continued increase in SA content in the bon1 mutant ( Figure 2 c).

[0204] In summary, the inventors found that SNC1 is involved in regulating plant growth under osmotic stress, and the dominant negative mutation of SNC1 can inhibit NLR signaling in the bon1 mutant and regulate plant growth under biotic and abiotic stresses.

[0205] Example 2: Calmodulin kinase CPK3 is involved in the response to osmotic stress

[0206] Compared with the wild type, the bon1 mutant has a phenotype of decreased calcium signal in the early response to osmotic stress. Therefore, the inventors measured the calcium signal of the screened ios mutant under osmotic stress to analyze whether the ios mutant can restore the phenotype of decreased calcium signal in the bon1 mutant under osmotic stress in addition to restoring the growth phenotype of the bon1 mutant. The results showed that compared with the bon1 mutant, ios could not restore the phenotype of decreased calcium signal in the bon1 mutant under osmotic stress ( Figure 4 a) The genetic relationship further shows that in response to osmotic stress, the protein encoded by the mutant gene of the ios mutant should be located downstream of the BON1 protein and after the change in calcium signal. Calcium signal is a conserved second messenger in plants and plays a role in signal transduction. The calcium decoding protein CPKs in plants binds to Ca 2+ After that, it will activate its kinase activity, perform signal regulation, and respond to various stress responses. It is an important regulatory factor for plant growth and survival.

[0207] Therefore, the inventors conducted plant tissue expression analysis on several major CPK proteins that regulate plant stress responses, including CPK3, CPK4, CPK5, CPK6, and CPK11. By searching the eFP (electronic fluorescent pictograph browser) database, they found that CPK3 ( Figure 4 b). In abiotic stress, cold stress and salt stress can both activate the kinase activity of calcium-dependent protein kinase CPK3. In order to explore whether osmotic stress can activate the kinase activity of CPK3, a transgenic material of CPK3proCPK3-4Myc / Col-0 was constructed. IP-CPK3 kinase and in vitro phosphorylation experiments showed that compared with non-treatment, osmotic stress treatment can significantly activate the kinase activity of CPK3 ( Figure 4 c).

[0208] In summary, osmotic stress can activate the kinase activity of CPK3, thereby transducing osmotic stress response signals.

[0209] Example 3: Sustained activation of CPK3 can inhibit NLR signaling to regulate plant growth

[0210] The constitutively activated form of CPK3 was transfected into the bon1 mutant using a transgenic method, thus constructing CPK3proCPK3 CA -4Myc / bon1-1 transgenic material. Previous experiments have shown that the bon1 mutant has a persistently activated NLR signal, which is manifested by cotyledon development defects under osmotic stress and an autoimmune dwarf phenotype in the soil. Therefore, the inventors investigated CPK3proCPK3 CA The phenotype of the transgenic materials of -4Myc / bon1-1 was analyzed and it was found that compared with bon1-1, CPK3proCPK3 CA -4Myc / bon1-1 transgenic materials can simultaneously restore the stunted cotyledon development under osmotic stress and the dwarf phenotype of immune self-activation in the soil of the bon1 mutant ( Figure 5 a and b). Further, CPK3proCPK3 CA -4Myc / bon1-1 transgenic materials can also restore the continuous high expression of NLR signal marker genes PR1 and PR2 in bon1 mutants under osmotic stress conditions ( Figure 5 c and d). At the same time, the transgenic material also restored the increase in SA content of the bon1 mutant under osmotic treatment ( Figure 5e). In summary, sustained activation of CPK3 can inhibit the hyperactivated NLR signaling in the bon1 mutant. This indicates that sustained activation of CPK3 can inhibit SNC1 and maintain plant growth.

[0211] Example 4. CPK3 and the TIR domain of TNL-like R protein SNC1 (SNC1 TIR )

[0212] It is known that SNC1, as a TNL-type R protein, has three domains, namely, the TIR domain, the NB-LRR domain and the LRR domain, and the TIR domain is mainly related to signal transduction. In order to further verify whether there is a biochemical connection between CPK3 and SNC1, this example explores the regulatory relationship between CPK3 and the TIR domain, that is, whether CPK3 interacts with the TIR domain.

[0213] Since the expression of TNL-TIR domain in wild-type tobacco leaves can cause hypersensitive response of local cell death in plants, the inventors conducted Co-IP and split-LUC experiments in the tobacco mutant eds1-2. The experimental results of Co-IP and split-LUC verified that CPK3 can interact with the TIR domain of SNC1 ( Figure 6 a and b).

[0214] Example 5: CPK3 phosphorylates the TIR domain of TNL-like R protein SNC1

[0215] This example investigates whether CPK3 can phosphorylate the TIR domain of SNC1.

[0216] The inventors purified GST-CPK3 and GST-SNC1 in vitro TIR ,pass 32 P-labeled in vitro phosphorylation assay confirmed that CPK3 can phosphorylate the TIR domain of SNC1. Figure 7 a) Further mass spectrometry analysis revealed that CPK3 can phosphorylate the S18 and S26 sites of the TIR domain of SNC1. Therefore, the inventors purified the inactive mutant GST-SNC1 of S18 and S26 in vitro. S18AS26A (GST-SNC1 TIRAA ), further through 32 In vitro phosphorylation assay with P-labeled GST-SNC1 TIR Compared with the mutant protein GST-SNC1 TIRAA The phosphorylation level of CPK3 was significantly reduced ( Figure 7a). For the S26 site of SNC1, the inventors prepared a phosphorylation antibody based on the DVFP-pS-FRGED peptide. Through protein immunoblotting analysis performed with the phosphorylation antibody of the S26 site, the inventors were able to verify that CPK3 can phosphorylate the S26 site of SNC1, and the phosphorylation level was significantly reduced after the addition of λ-protein phosphatase (λ-PPase) ( Figure 7 b) In summary, CPK3 can indeed phosphorylate SNC1 TIR The S18 and S26 sites.

[0217] Example 6: Osmotic stress-induced CPK3 can phosphorylate SNC1 TIR

[0218] The experimental results of Examples 1 to 5 have verified that both CPK3 and SNC1 can regulate plant growth under osmotic stress, and osmotic stress can activate the kinase activity of CPK3. Biochemical experiments have further verified that CPK3 can directly regulate SNC1. TIR , and whether osmotically activated CPK3 can phosphorylate SNC1 TIR This is the next question the inventor wants to explore.

[0219] Therefore, the inventors treated the transgenic material of CPK3proCPK3-4Myc / Col-0 under osmotic stress and non-osmotic stress, and then enriched CPK3 by IP. TIR Phosphorylation experiments revealed that IP-CPK3 was significantly increased for SNC1 after osmotic stress compared with non-osmotic stress. TIR The phosphorylation of Figure 8 ).

[0220] In summary, the CPK3 kinase activity induced by osmotic stress can further phosphorylate SNC1 TIR .

[0221] Example 7: The S26 site is highly conserved in plants, bacteria and mammals

[0222] The in vitro phosphorylation experiment in Example 5 has verified that CPK3 can phosphorylate SNC1 TIR The S18 and S26 sites in the genus were analyzed for their conservation, and it was found that the S26 site was highly conserved. The inventors compared the sequences of 141 proteins with TIR domains in Arabidopsis thaliana and found that the S26 site was highly conserved ( Fig. 9 a). The TIR domain of Arabidopsis is also conserved in other plant species. The inventors selected the L6 from flax, which has received much attention. TIRProtein comparison revealed that L6 TIR The S26 position is also conserved ( Fig. 9 a) At the same time, the plant TIR domain is conserved in bacteria and mammals. The inventors have studied the bacterial Ab protein and ThsB with TIR domain and the human hSARM1 TIR (UniProtKB / Swiss-Prot:Q6SZW1.1) protein sequence alignment revealed that the S26 site was also conserved ( Fig. 9 a). Therefore, the inventors further verified the reliability of the conservation of the S26 site through in vitro phosphorylation experiments. The inventors purified His-L6 in a prokaryotic system. TIR and conserved site-specific knockout protein His-L6 TIRS66A Protein, through 32 In vitro phosphorylation assay with P labeling, compared with wild type L6 TIRS66A The phosphorylation level of CPK3 was significantly reduced ( Fig. 9 b). RBA1 is a TIR-only protein in Arabidopsis. The inventors purified the RBA1-Flag protein in an insect purification system and verified that the RBA1-Flag protein can be phosphorylated by CPK3 through an antibody-labeled in vitro phosphorylation experiment ( Fig. 9 c) The inventors also purified hSARM1 in a prokaryotic system. TIR hSARM1 TIRS567A Protein, through 32 In vitro phosphorylation assays labeled with P showed that hSARM1 TIRS567A The phosphorylation level of the protein by AtCPK3 was significantly reduced ( Fig. 9 d).

[0223] In summary, the S26 site of SNC1 is highly conserved in plants and mammals, and the conservation of this phosphorylation and phosphorylation site was verified through a series of experiments.

[0224] Example 8: AtCPK3 phosphorylates the TIR domain of RPS4

[0225] The phosphorylated S26 site is highly conserved in plants, bacteria, and mammals, but there are still TNL proteins with non-conserved S26 sites in Arabidopsis, one of which is Pair-TNL, such as RPS4-RRS1 and CHS3-CSA1 ( Fig. 9 a).

[0226] The inventors then explored whether AtCPK3 would phosphorylate this type of Pair-TNL. The inventors purified His-RPS4 in a prokaryotic expression system. TIR , and carried out 32P-labeled in vitro phosphorylation assay revealed that AtCPK3 can phosphorylate RPS4 TIR ( Fig.10 The above results suggest that although the S26 site is not conserved in some special types of Arabidopsis TNL-like R proteins, CPK3 can also phosphorylate the TIR domains of these special types of TNL-like R proteins.

[0227] In summary, the regulatory mechanism of AtCPK3 phosphorylation of TNL-TIR is highly conserved in Arabidopsis.

[0228] Example 9: CPK4 / 5 / 6 / 11 phosphorylates SNC1 TIR Regulation of plant growth under osmotic stress

[0229] The CPKs in Arabidopsis are a very large family, with a total of 34 CPK kinases. Although the inventors already knew that CPK3 can phosphorylate SNC1 TIR , but do other CPKs phosphorylate SNC1 TIR This is the next question the inventor explored.

[0230] The inventors selected some classic CPK proteins in the research, such as CPK5, CPK4, CPK6 and CPK11. 32 The P-labeled in vitro phosphorylation assay verified that typical CPK4, CPK5, CPK6, and CPK11 can phosphorylate SNC1. TIR ( Fig.11 a and b). Taking CPK5 as an example, it is related to wild-type SNC1 TIR Compared with the inactivating mutant SNC1 TIRAA Phosphorylation levels by CPK5 were significantly reduced, suggesting that different CPKs phosphorylate SNC1 TIR The phosphorylation sites are also conserved ( Fig.11 a). At the same time, the inventors conducted phenotypic analysis on the cpk4 / 5 / 6 / 11 quadruple mutant and found that under osmotic stress, the quadruple mutant showed a typical phenotype of delayed rosette leaf development ( Fig.11 c).

[0231] In summary, multiple CPKs phosphorylate SNC1 in Arabidopsis TIR The mechanism is conserved, and multiple CPKs are also required for regulating plant growth under osmotic stress.

[0232] Example 10: Phosphorylated serine sites affect the function of glutamate sites in catalysis at the structural level

[0233] TNL-TIR in plants, mammals and some bacteria can enzymatically degrade NAD +In mammals, hSARM1 TIR Can enzymatically hydrolyze NAD + Produces cADPR; in plants, TNL-TIR digests NAD + The cADPR variant, v-cADPR, is produced. Whether it is plant or mammalian TNL-TIR proteins or some bacteria, their NAD + The enzyme activity depends on a conserved glutamate site. TIR and hSARM1 TIR Structural analysis revealed that the hydroxyl group at the conserved serine site and the carboxyl group at the catalytic glutamate site are very close in structure ( Fig.12 ). + Before enzymatic hydrolysis, the catalytic glutamate site will bind to the substrate to form an intermediate product of glycosyl-enzyme. The phosphorylated serine site will be closer to the glutamate site in terms of structure, and the phosphorylated serine site will also carry a negative charge, causing the phosphorylated serine site to repel the catalytic glutamate site at the structural level, thereby hindering the glutamate site from binding to the substrate to form an intermediate product of glycosyl-enzyme, affecting the enzymatic hydrolysis of NAD by TNL-TIR. + enzyme activity and product production.

[0234] Example 11: Phosphorylated serine sites affect the formation of TIR domain oligomers

[0235] TNL-TIR forms a TIR-TIR oligomer structure, which is the prerequisite for its function. + The degradation function of TIR depends on the formation of dimers, so the inventors wanted to verify whether the phosphorylation of TIR domain would affect the formation of oligomers.

[0236] L6 TIR Taking RBA1 and wild-type TIR as examples, yeast two-hybrid experiments were performed. Both wild-type TIR and simulated phosphorylation inactivation mutant TIRSA detected TIR-TIR interactions in the yeast system, i.e., the formation of TIR-TIR oligomers. However, the mutant TIRSD, which simulates continuous phosphorylation, did not detect interactions in the yeast system, i.e., it could not form oligomers ( Fig.13 a and b). The yeast two-hybrid results also suggest that phosphorylation regulation can also affect the formation of TIR-TIR oligomers, thereby affecting TNL-TIR including NAD + Various biological functions including enzyme activity.

[0237] Example 12: RBA1 can enzymatically hydrolyze NAD +Generation of Nam and 2'cADPR

[0238] Phosphorylation of serine sites affects the binding of TNL-TIR to NAD + The glycosyl-enzyme structure formed by the combination will also affect the formation of TIR-TIR dimerization and affect its biological function. Therefore, in this embodiment, the inventors want to explore whether the phosphorylated serine site will really affect its degradation of NAD + level.

[0239] First, NAD + , Nam and cADPR standards were subjected to mass spectrometry to determine their respective retention times on the mass spectrometer ( Fig.14 a, c and e). Although TNL-TIR in plants can degrade NAD + , but most plant TNL-TIRs degrade NAD + The enzyme activity of RBA1 is difficult to measure. Therefore, the inventors selected RBA1 protein with relatively high enzyme activity to establish the system. The inventors first purified RBA1-Flag and glutamate site inactivation mutation RBA1E86A-Flag in the insect purification system, and then + The incubation was performed and the reaction solution was then subjected to mass spectrometry. Compared with the wild type, the glutamate inactivation mutation degraded NAD + The ability to Fig.14 b). At the same time, compared with the wild type, the value of Nam produced by RBA1E86A-Flag in the reaction solution was also significantly reduced ( Fig.13 d). Because the standard substance uses cADPR, and RBA1 produces 2'cADPR, there is some deviation in the peak retention time of the two on the mass spectrometer ( Fig.14 e). According to the retention time of 2'cADPR, the 2'cADPR produced by RBA1E86A-Flag reaction was significantly reduced compared with the wild type ( Fig.14 f).

[0240] In summary, RBA1 can degrade NAD + , producing Nam and 2'cADPR. It also shows that the highly conserved glutamate site is indeed very important for the enzymatic activity of RBA1.

[0241] Example 13: In vitro enzyme activity experiments prove that phosphorylation inhibits the enzyme activity and product production of RBA1

[0242] Through mass spectrometry and in vitro enzyme activity experiments, the inventors established a determination system for RBA1 enzyme activity and determined that NAD +The next step is to verify whether phosphorylation affects the enzyme activity of RBA1 and the production of its products.

[0243] The wild-type RBA1-Flag protein, the RBA1S19A-Flag protein simulating point inactivation, and the RBA1SD-Flag protein simulating sustained phosphorylation were expressed and purified by the insect expression and purification system. The enzyme activity reaction was carried out by an in vitro enzyme activity system, and then the mass spectrometry of the substrate and the product was determined. Through mass spectrometry analysis, the inventors found that the protein RBA1S19D simulating sustained phosphorylation degraded NAD + The activity is much lower than that of the wild-type protein ( Fig.15 a and b). At the same time, the production of Nam and 2'cADPR by RBA1S19D-Flag protein simulating sustained phosphorylation was much lower than that of wild type ( Fig.15 c, d, e and f). At the same time, the inventors found that the protein RBA1SA-Flag, which simulates the inactivation of the spike, degrades NAD + Although the activity is lower than that of the wild type, it is much higher than that of the RBA1S19D-Flag protein that simulates sustained phosphorylation ( Fig.15 a and b), the production of products Nam and 2'cADPR is also between the wild type and RBA1S19D-Flag ( Fig.15 c, d, e and f). At the same time, the amount of the reacting RBA1-Flag protein, RBA1S19A-Flag protein and RBA1S19D-Flag protein was quantified using Anti-Flag antibody ( Fig.15 g).

[0244] In summary, the in vitro enzyme activity reaction experiment shows that the phosphorylation of RBA1 inhibits the proteolysis of NAD by RBA1 + The enzyme activity and the production of Nam and 2'cADPR products.

[0245] Example 14: RBA1 phosphorylation inhibits the accumulation of 2'cADPR in plants

[0246] The in vitro enzyme activity experiment of Example 13 demonstrated that phosphorylation of RBA1 can inhibit its enzyme activity, so the inventors wanted to verify whether the same phenomenon occurs in plants.

[0247] Overexpression of TNL-TIR in tobacco leaves induces localized cell death in plants and is NAD-dependent +Enzyme activity. Before causing local cell death, 2'cADPR will also accumulate in large quantities in the body. The activity of the TIR domain depends on the downstream key node EDS1, which is also crucial for local cell death caused by TIR. Therefore, the inventors used the accumulation of 2'cADPR in the body as an indicator, expressed RBA1-YFP, RBA1S19A-YFP, RBA1S16D-YFP and the highly conserved glutamate inactivating mutant RBA1E86A-YFP in the leaves of the eds1-2 tobacco mutant, and then performed mass spectrometry measurements. Two days after the injection, the tobacco leaves were extracted and mass spectrometrically determined, and it was found that the accumulation of 2'cADPR in the tobacco leaves injected with RBA1S19D-YFP and RBA1E86A-YFP was much lower than that in the tobacco leaves injected with RBA1-YFP and RBA1S19A-YFP ( Fig.16 a). At the same time, the inventors used Anti-GFP antibody to quantify the expression of wild-type and mutant RBA1-YFP and found that the protein expression levels of wild-type and mutant samples used for mass spectrometry were basically the same ( Fig.16 b).

[0248] In summary, RBA1 phosphorylation inhibits NAD + Enzyme activity-dependent accumulation of 2'cADPR.

[0249] Example 15: CPK3CA inhibits TNL-TIR-induced cell death

[0250] The inventors have previously demonstrated through in vivo and in vitro experiments that phosphorylated RBA1 inhibits its NAD + The activity of the enzyme further affects the production of its products. + Enzyme activity is the prerequisite for the hypersensitive phenotype of local death in tobacco leaves. Therefore, the inventors wanted to explore whether phosphorylation would affect cell death in tobacco leaves induced by TNL-TIR. Because the expression abundance of CPK3CA (a truncated sequence in the CPK3 coding region, encoding positions 1-341 of the CPK3 protein) is not very stable in tobacco leaves, the inventors chose the CPK3CA-cLUC-Flag vector for the experiment after trial and error.

[0251] The inventors co-expressed CPK3CA-cLUC-Flag with some plant TNL-TIR proteins, such as L6 TIR -YFP, SNC1 TIR -YFP and RBA1-HA, while the single expressed TNL-TIR was used as a control. Because different TNL-TIR induced different degrees of cell death, the inventors classified and labeled them with three different degrees of death ( Fig.17a). The inventors can know from the results that L6 TIR -YFP, SNC1 TIR -YFP and RBA1-HA can cause obvious cell death phenotype in tobacco leaves, but this hypersensitive phenotype was significantly inhibited after co-expression of CPK3CA-cLUC-Flag ( Fig.17 b). By detecting the expression level with Anti-GFP and Anti-Flag antibodies, it was found that co-expression did not cause obvious changes in abundance ( Fig.18 ).

[0252] In summary, CPK3CA inhibits TNL-TIR-induced cell death.

[0253] Example 16, SNC1 TIR Phosphorylation inhibits cell death induction

[0254] Co-expression of TNL-TIR and CPK3CA inhibited the local death of tobacco leaves induced by TNL-TIR. The inventors then wanted to explore whether the phenotype was inhibited by phosphorylation of TIR.

[0255] The inventors first injected SNC1 into wild-type tobacco leaves TIR -YFP, SNC1 TIRS26A -YFP, SNC1 TIRS26D -YFP, SNC1 TIRS18A -YFP, SNC1 TIRS18D -YFP, SNC1 TIRS18AS26A(AA) -YFP, SNC1 TIRS18D / S26D(DD) -YFP and SNC1 TIRE84A -YFP. According to previous reports, SNC1 TIRE84A -YFP was used as a phenotypic control. Because the phenotype of the S18 site related mutation is not easy to observe, the inventors selected different concentrations of Agrobacterium for injection, OD = 0.2 and OD = 0.4 respectively. The above expression samples were sampled and analyzed by Western blot two days after injection, and photographed and observed three days later. The photography was carried out under normal light and ultraviolet light respectively to accurately determine the degree of cell death, and the number and degree of death of the tobacco leaves after injection were statistically analyzed in the form of a stacking diagram.

[0256] Through observation and statistics, it can be seen that injection of SNC1 TIRS18D -YFP, SNC1 TIRS26D -YFP and SNC1 TIRS18D / S26D(DD The extent of local cell death in tobacco leaves injected with )-YFP was significantly lower than that injected with SNC1 TIRWT 、SNC1 TIRS18A 、SNC1 TIRS26Aand SNC1 TIRS18AS26A(AA) -YFP in tobacco leaves and with the glutamate-inactivating mutant SNC1 TIRE84A -YFP injected leaves have the same phenotype ( Fig.19 ac). At the same time, although SNC1 TIRS18D -YFP and SNC1 TIRS26D -YFP mutations have an effect, but the local death phenotype of tobacco leaves induced by the mutation simulating sustained phosphorylation at the S26 site is significantly lower than the phenotype induced by the mutation simulating sustained phosphorylation at the S18 site. This shows that although both the S18 site and the S26 site can be phosphorylated and play a certain role, the biological significance of the conservative S26 site is more significant when combined with the phenotypic and conservative analysis. At the same time, the simulated inactivation mutation SNC1 at the S26 site TIRS26A The phenotype is intermediate between wild-type and simulated SNC1 phosphorylation TIRS26D The phenotypes between Fig.19 a), this experimental result is consistent with the results of the previous enzyme activity experiment. Samples were taken two days after injection and Western blot analysis showed that wild-type SNC1 TIR Different mutant forms of SNC1 TIR The protein expression level did not change significantly within two days, indicating that phosphorylation-inhibited SNC1 TIR The phenotype is not related to protein expression level in the short term ( Fig. 20 ).

[0257] In summary, the experiments show that phosphorylated SNC1 TIR It will inhibit the local cell death phenotype induced by it in tobacco leaves.

[0258] Example 17 Phosphorylation of plant TNL-TIR inhibits its induced cell death

[0259] Thanks to SNC1 TIR The S26 site is highly conserved in most plants. This example further explores whether phosphorylation also inhibits the local death phenotype of tobacco leaves induced by TNL-TIR in other plants.

[0260] The inventor selected L6, which has received relatively high attention. TIR The experiment was conducted by injecting L6 into tobacco leaves to express TIRWT / S66A / S66D / E135A -YFP and RBA1 WT / S19A / S19D / E86A-HA, similarly, samples were taken for protein expression analysis two days after injection, and phenotype photography was performed three days after injection. TIRWT -YFP and RBA1 WT-HA compared, L6 TIRS66D-YFP and RBA1S19D-HA, like the inactivation mutation of the glutamate active site, significantly reduced the level of localized death induced by tobacco leaves. TIRS66A The cell death phenotypes of -YFP and RBA1S19A-HA were between WT and SD ( Fig.21 a and b). Meanwhile, Western blotting analysis revealed that L6 TIRS66D -YFP expression levels were consistently lower than those of wild type and other mutants, suggesting that L6 TIR Phosphorylation may be associated with protein degradation pathways ( Fig. 22 a).

[0261] In summary, phosphorylation of TNL-TIR in most plants will inhibit the cell death phenotype induced by it.

[0262] Example 18 Phosphorylation of bacterial and mammalian TIR inhibits TIR-induced cell death

[0263] SNC1 TIR The S26 site of is highly conserved in bacterial and mammalian TNL-TIRs and is phosphorylated by AtCPK3. Therefore, this example further explores whether bacterial and mammalian TNL-TIR phosphorylation can also inhibit the local death phenotype of tobacco leaves induced by them.

[0264] The inventors selected a protein with relatively large number of reports and NAD + Enzymatically active TIR protein, hSARM1 in mammals TIR Abs in proteins and bacteria TIR hSARM1 was expressed by injection into tobacco leaves TIRWT / S567A / S567D / E642A -YFP and Ab TIRWT / S140A / S140D / E208A -YFP experiment, samples were taken two days after injection for protein expression analysis and phenotype photography. TIRWT -YFP and Ab TIRWT -YFP compared to hSARM1 TIRS567D -YFP and Ab TIRS140D -YFP and control hSARM1 TIRE642A -YFP and Ab TIRE208A -YFP significantly reduced the degree of cell death in tobacco leaves; however, hSARM1 TIRS567A -YFP and Ab TIRS140A -YFP is intermediate between wild-type and mimic phosphorylated TIR phenotypes ( Fig.23 a and b). Analysis of the expression level of the injected protein by Anti-GFP antibody showed that the protein expression levels between the wild type and various mutants were basically consistent ( Fig.24 ).

[0265] In summary, phosphorylation of TNL-TIR proteins in plants, animals and bacteria will inhibit the phenotype of cell death induced by them. Similarly, it suggests that phosphorylation of TNL-TIR proteins in plants, animals and bacteria will inhibit their biochemical functions and activities.

[0266] Example 19: Phosphorylated TIRs regulate and maintain plant growth

[0267] In this example, the inventors wanted to further explore whether phosphorylated TIRs regulate and maintain plant growth.

[0268] Since overexpression of full-length NLR protein in plants can cause the autoimmune phenotype of dwarfed plants, the inventors constructed and obtained the transgenic material of OE-SNC1 / Col-0. It was found that the transgenic material of OE-SNC1 / Col-0 can indeed cause the autoimmune phenotype of plants, such as dwarfed growth and continuously elevated SA content in the body ( Fig.25 a and e). At the same time, the transgenic material of OE-SNC1DD / Col-0 was constructed and analyzed by phenotypic observation, PR1 / PR2 marker gene detection and determination of SA content in vivo. Compared with the transgenic material of OE-SNC1 / Col-0, the transgenic material of OE-SNC1DD / Col-0 restored the short autoimmune phenotype induced by overexpression of full-length TNL ( Fig.25 a). Like the bon1 mutant, the OE-SNC1 / Col-0 transgenic material exhibits a developmental retardation phenotype under osmotic stress ( Fig.25 b). On the other hand, compared with the transgenic material of OE-SNC1 / Col-0, the transgenic material of OE-SNC1DD / Col-0 did not show obvious developmental retardation phenotype under osmotic stress treatment ( Fig.25 b). At the same time, the expression of immune stress marker genes PR1 and PR2 induced by osmotic stress was significantly increased in the transgenic materials of OE-SNC1 / Col-0. Compared with the transgenic materials of OE-SNC1 / Col-0, the expression of PR1 / PR2 in the transgenic materials of OE-SNC1DD / Col-0, which simulates the persistent phosphorylation mutation, was significantly reduced under osmotic stress treatment ( Fig.25 c and d). Osmotic stress induces an increase in the SA content in plants. At the same time, compared with OE-SNC1 / Col-0, the SA content induced by the transgenic material of OE-SNC1DD / Col-0 under osmotic stress was significantly reduced ( Fig.25 e).

[0269] In summary, phosphorylation modification of TIRs is involved in regulating and maintaining plant growth.

[0270] Example 20: Phosphorylated TIRs partially inhibit plant immune regulation and plant growth

[0271] This example further explores whether phosphorylated TIRs can regulate plant immunity.

[0272] The transgenic materials of OE-SNC1 / Col-0 and OE-SNC1DD / Col-0 were infected with the pathogen pstDC3000. It was found that the transgenic materials of OE-SNC1 / Col-0 showed strong resistance. Unexpectedly, the transgenic materials of OE-SNC1DD / Col-0 also showed a certain resistance compared with Col-0 ( Fig.26 a and b). Infection with the pathogen pstDC3000 can induce a significant increase in the SA content in plants. Compared with the transgenic material of OE-SNC1 / Col-0 and Col-0, the SA content of the transgenic material of OE-SNC1DD after pstDC3000 infection was between the two materials ( Fig.26 c), which also indicates that the transgenic material of OE-SNC1DD / Col-0 has resistance to pathogens.

[0273] In summary, phosphorylated TNL-TIRs can regulate plant growth under biotic and abiotic stresses, and on the other hand, can also give plants a certain degree of disease resistance. This phosphorylation regulation has application prospects in breeding.

[0274] discuss

[0275] The present invention describes that calcium signal-activated CPK3 can phosphorylate a highly conserved serine site of TIR-NLRs, and the degradation of the phosphorylated TIR domain NAD + The enzyme activity of CPK3 is inhibited, thereby regulating the growth of plants during the immune process and stress response. The continuously activated form of CPK3 can restore the growth defect phenotype of plants under stress conditions caused by the activation of TIR-NLRs. The mutant form TIRSD that mimics the sustained phosphorylation can inhibit the TIR domain from activating NAD. + The degradation of 2'cADPR inhibits the production of products such as 2'cADPR, resulting in the loss of its function of inducing cell death phenotype.

[0276] Osmotic stress is a typical early signal of abiotic stress, such as drought and salt stress. Under long-term osmotic stress, the expression of immune downstream marker genes PR1 / PR2 can be induced, and the SA level in the plant can also be increased, inhibiting the growth of plants under stress conditions. The CPK3-TIRs phosphorylation regulatory module can inhibit the increase of PR1 / PR2 induced by osmotic conditions in plants and the accumulation of SA content, and promote the growth of plants under osmotic stress conditions. Overexpression of TNLs can lead to plant autoimmune growth defects and growth defects in osmotic stress treatment, while inducing a continuous increase of PR1 / PR2 and accumulation of SA content in plants. TNLSD, which simulates continuous phosphorylation, can restore the defects of plant autoimmune phenotypes, reduce the increase of PR1 / PR2 and SA accumulation induced by osmotic stress, and give plants a certain disease resistance, balancing the growth and resistance of plants under stress conditions. Plant TNLs proteins are intracellular receptors for effector proteins released when pathogens infect plants, and the TIR domain, as the N-terminal domain of TNLs proteins, is mainly involved in signal transduction. Studies have found that the TIR domain has a typical NAD + AtCPK3 phosphorylates TIRs, which can regulate the biochemical characteristics of enzymes and DNA / RNA enzymes, but little is known about the molecular regulatory mechanism of TIR domains, especially post-translational protein modification regulation. AtCPK3 phosphorylates TIRs to fill the gap in the field of protein modification regulation of TIR domains, and analyzes how plants balance the relationship between resistance and growth under stress, including biotic and abiotic stresses. As a key node in regulating plant growth, the phosphorylation regulatory module of CPK3-TIR has certain reference significance for cultivating new crop varieties with good stress resistance and both yield and growth.

[0277] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims

1. A method for regulating plant immunity, comprising: Regulate the CPKs-TIR-NLRs signaling pathway or its pathway molecules in the plant.

2. The method according to claim 1, characterized in that Regulating CPKs-TIR-NLRs or their pathway molecules, thereby regulating plant immunity; the regulation of plant immunity includes: enhancing the plant's ability to respond to stress or its tolerance to stress, inhibiting plant cell death, and promoting plant growth.

3. The method according to claim 2, characterized in that The regulation of the CPKs-TIR-NLRs signaling pathway or its pathway molecules includes: increasing the phosphorylation level of TIR, promoting CPKs-TIR interaction to inhibit TIR, and regulating the immune ability of the plant; preferably, increasing the phosphorylation level of TIR includes: increasing the phosphorylation level of Ser at position 26 and / or position 18 of TIR; preferably, simulating phosphorylation modification by mutating Ser to Asp.

4. The method according to any one of claims 1 to 3, characterized in that: The regulation includes: enhancing the ability of CPKs to phosphorylate TIR; transferring the coding gene of CPKs or the expression construct or vector containing the coding gene into the plant; activating CPKs with calcium ions; performing gain-of-function mutation on CPKs; promoting the expression of CPKs with an expression-enhancing promoter or a tissue-specific promoter; or promoting the expression of CPKs with an enhancer; Preferably, the CPKs include CPK3, CPK4, CPK5, CPK6, CPK11; and / or, Preferably, the TIR comprises: a SNC1 protein, a TNLs protein or a NLRs protein containing a TIR domain.

5. Use of the CPKs-TIR-NLRs signaling pathway, its pathway molecules or their regulators for regulating the immune capacity of plants; preferably, the CPKs include CPK3, CPK4, CPK5, CPK6 and / or CPK11.

6. The use according to claim 5, characterized in that The regulator is used to regulate plant immunity; regulating plant immunity includes: enhancing the plant's ability to respond to stress or its tolerance to stress, inhibiting plant cell death, and promoting plant growth; Preferably, the regulator includes: an agent that increases the phosphorylation level of TIR, and an agent that promotes CPKs-TIR interaction to inhibit TIR; preferably, the agent that increases the phosphorylation level of TIR includes: an agent that increases the phosphorylation level of Ser at position 26 and / or position 18 of TIR, and more preferably includes an agent that mutates Ser to Asp; Preferably, the regulator includes: a CPKs upregulator; more preferably includes: an exogenous CPKs encoding gene or an expression construct or vector containing the encoding gene, calcium ions, or an agent for performing a gain-of-function point mutation on CPKs; Preferably, the CPKs include CPK3, CPK4, CPK5, CPK6 and / or CPK11.

7. The use according to claim 5, characterized in that The stress includes osmotic stress; preferably, the osmotic stress includes abiotic stress; preferably, the abiotic stress includes: salt stress, cold stress, drought stress.

8. A method for identifying plant immunity, comprising: Analyze the accumulation level of phosphorylated proteins and protein phosphorylation levels of TIR domains in plants; If the accumulation level of phosphorylated proteins in the TIR domain and / or the protein phosphorylation level is normal, the plant immune ability is normal; if the accumulation level of phosphorylated proteins in the TIR domain and / or the protein phosphorylation level is abnormal, the plant immune ability is abnormal; Preferably, analyzing the phosphorylation level of the protein comprises: analyzing the phosphorylation level of Ser at position 26 and / or Ser at position 18 of TIR; The plant immunity includes: the ability to respond to osmotic stress or the tolerance to stress, the survival of plant cells, and the growth of plants.

9. A method for screening a substance for regulating plant immunity, comprising: (1) adding the candidate substance to a system expressing the CPKs-TIR-NLRs signaling pathway or its pathway molecules; (2) detecting the system and observing the phosphorylation level of TIR therein. If the phosphorylation level is increased, it indicates that the candidate substance is a substance that can be used to regulate plant immunity; Preferably, the step of increasing the phosphorylation level of TIR comprises: increasing the phosphorylation level of Ser at position 26 and / or Ser at position 18 of TIR; Preferably, the CPKs include CPK3, CPK4, CPK5, CPK6 and / or CPK11; Preferably, the TIR comprises: a SNC1 protein, a TNLs protein or a NLRs protein containing a TIR domain.

10. The method according to claim 9, characterized in that (2), including: observing the effect of the candidate substance on the interaction between CPKs and TIR; if it promotes the interaction between the two and promotes CPKs to phosphorylate TIR, it indicates that the candidate substance is a substance that can be used to regulate plant immunity; preferably, the CPKs phosphorylation of TIR includes: phosphorylating Ser at position 26 and / or position 18 of TIR.

11. A use of a plant CPKs-TIR-NLRs signaling pathway or its pathway molecules as a molecular marker for identifying the immune capacity of a plant; Preferably, the CPKs include CPK3, CPK4, CPK5, CPK6 and / or CPK11.

12. The method or use according to any one of claims 1 to 11, characterized in that: The plants are selected from the group consisting of: Plants containing CPKs-TIR-NLRs signaling pathway; dicotyledons; Linaceae; Cruciferous vegetables; Preferably, the dicotyledonous plants include soybeans; the flaxaceae plants include flax; the cruciferous plants include Arabidopsis thaliana, Chinese cabbage, kale, mustard, radish, and rapeseed.

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