Blue light control system containing lurp1 and its application in activating plant immune capacity

Abstract

The application provides a blue light control system containing LURP1 and application thereof in activating plant immune capacity. The application newly finds a gene LURP1 which is simultaneously induced by blue light and pathogenic bacteria and positively regulates plant immune capacity, and the LURP1 constitutes a new blue light control system based on blue light together with upstream and downstream molecules (including CRY1 and FLS2). The LURP1 can be palmitoylated to a cell membrane after pathogen invasion, and activates an important receptor kinase FLS2 in PTI. The technical scheme of the application is helpful for molecular design breeding, and provides a theoretical basis for cultivating high-quality varieties which can resist pathogen invasion and also consider yield.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and botany, and more specifically, this invention relates to a blue light control system containing LURP1 and its application in activating plant immunity. Background Technology

[0002] Light is a crucial factor influencing plant growth and development. Not only is light the primary energy source for photosynthesis, but it also acts as a signaling molecule, regulating various aspects of plant growth and development, such as hypocotyl elongation, flowering, the biological clock, stomatal development, and the signal transduction of various hormones. The perception of light signals is primarily accomplished by several types of photoreceptors, including known red / far-red light receptors (phyA, phyB, phyC, phyD, phyE), blue / UVA light receptors (CRY1, CRY2, PHOT1, PHOT2, ZTL, FKF, LKP2), and the UV-B receptor UVR8. These receptors can sense and transmit light signals, thereby regulating plant growth and development. Cryptochrome (CRY), a blue light receptor, is the only known photoreceptor that is conserved across bacteria, yeast, insects, mammals, and higher plants. In bacteria, CRY mediates DNA repair; in animals, it is a core member of the biological clock; and in higher plants, CRY acts as a photoreceptor, mediating light signals to regulate plant growth, development, flowering, the biological clock, and the signal transduction of various hormones.

[0003] Plant immune responses primarily follow two modes. One is pathogen-associated molecular pattern-triggered immunity (PTI), where PTI initiates signal transduction based on receptors or receptor-like proteins on the cell membrane, thereby activating the expression of resistance genes. Although PTI can resist infection by most pathogens, with the evolution of pathogens, some pathogens still manage to bypass PTI and infect plants. Therefore, plants have evolved another defense mechanism called effector-triggered immunity (ETI). ETI elicits a stronger immune response, mainly through the specific recognition of effector factors secreted by pathogens by NB-LRR receptors, thereby further blocking pathogen invasion. During PTI, the plant receptor-like kinase FLS2 recognizes the 22 conserved amino acids (Flg22, Flagellin 22) at the N-terminus of bacterial flagellin. After recognizing flg22, FLS2 rapidly binds to and co-phosphorylates and activates the receptor BAK1. The phosphorylated and activated BAK1 then phosphorylates another important membrane-localized kinase, BIK1 (Botrytis-Induced Kinase 1). BIK1, in turn, phosphorylates the FLS2-BAK1 complex. Finally, the activated BIK1 dissociates from the receptor complex and activates downstream intracellular signaling. Downstream, the MPK cascade signaling is a crucial component of PTI. A typical MPK cascade signaling consists of sequentially phosphorylated MAPKKKs, MAPKKs, and MAPKs, which amplify and activate downstream transcription factors, initiating plant immune regulation.

[0004] Light signals play a crucial role in plant resistance to pathogens. As early as 1970, research indicated that light enhances plant resistance. Subsequent studies have shown that tobacco (Nicotiana tubacum) resists *Pseudomonas solanacearum*, rice (Oryza sativa) resists *Xanthomonas oryzae*, broad bean (Vicia faba L.) resists *Botrytis cinerea*, and Arabidopsis thaliana resists *Peronospora parasitica* and *Pseudomonas syringae*—all of which are light-dependent. Photoreceptors also play important roles in resisting pathogen stress; the red light receptor double mutant phyA phyB and the blue light receptor mutant cry1 are both more susceptible to *P. DC3000* bacteria. In 2010, Wu and Yang reported that CRY1 participates in the positive regulation of R protein-mediated resistance to *P. syringae*.

[0005] However, how light regulates the immune (disease resistance) response of plants and what the underlying molecular mechanisms are have not been reported in this field to date. Summary of the Invention

[0006] The purpose of this invention is to provide a blue light control system containing LURP1 and its application in activating plant immunity.

[0007] In another aspect of the invention, a method for enhancing plant immunity is provided, comprising: upregulating (e.g., promoting / activating / enhancing) a blue light-based light control system in the plant; said light control system comprising CRY1 and its interacting protein LURP1; said CRY1 activating LURP1 under blue light, said LURP1 being localized to the cell membrane (induced by pathogen infection of the plant), thereby enhancing plant immunity.

[0008] In one or more embodiments, the light control system further includes a receptor-like kinase FLS2, forming the CRY1-LURP1-FLS2 pathway.

[0009] In one or more embodiments, CRY1 activates LURP1 under blue light. When plants are infected by pathogens, LURP1 localizes to the cell membrane, binds to receptor kinase FLS2, and enhances the immune activation activity of FLS2, thereby enhancing the plant's immunity.

[0010] In one or more embodiments, when a plant is infected by a pathogen, the LURP1 undergoes N-terminal palmitoylation and is localized to the cell membrane; preferably, the cysteine ​​residue at position 13 undergoes palmitoylation.

[0011] In one or more embodiments, the light control system further includes BAK1, wherein FLS2 and BAK1 form an FLS2-BAK1 immune complex (i.e., form the CRY1-LURP1-FLS2-BAK1 pathway), and LURP1 promotes the formation of immune complexes and specific membrane localization of FLS2 and BAK1, enhances the activity of FLS2-BAK1, activates downstream molecules, and enhances the plant's immunity.

[0012] In one or more embodiments, the blue light-based light control system operates in an environment where blue light (including low or high blue light intensity) is present.

[0013] In one or more embodiments, the blue light is in the range of 1–200 μmol·m⁻¹. -2 ·s -1 Blue light; preferably 2–150 μmol·m –2 ·s –1 Blue light; preferably 3–100 μmol·m –2 ·s–1 Blue light; such as 5, 10, 15, 20, 25, 30, 50, 80, 100, 120 μmol·m –2 ·s –1 Blue light.

[0014] In one or more embodiments, the enhancement of plant immunity includes: enhancing the plant's disease resistance (ability to resist pathogens); preferably, the pathogen is a plant pathogen (such as plant pathogenic bacteria).

[0015] In one or more embodiments, the pathogen includes: a pathogen that induces / triggers activation of the blue light-based light control system or molecules in the system after infecting a plant.

[0016] In one or more embodiments, the pathogen includes bacteria, fungi, viruses, viroids, etc.

[0017] In one or more embodiments, the pathogen comprises: bacterial flagellin or its N-terminus conserved 22 amino acids (Flg22, Flagellin 22).

[0018] In one or more embodiments, the light control system is a separate light control system.

[0019] In one or more embodiments, the CRY1 and / or LURP1 includes recombinantly expressed (overexpressed) CRY1 and / or LURP1.

[0020] In one or more embodiments, the upregulation of blue light-based light control systems in plants includes: increasing LURP1 expression, increasing CRY1 expression, promoting CRY1 activation of LURP1, promoting N-terminal palmitoylation of LURP1, promoting LURP1 localization to the cell membrane, or promoting the interaction between LURP1 and FLS2 to enhance the immune activation activity of FLS2.

[0021] In one or more embodiments, the enhancement of LURP1 expression includes introducing the LURP1 coding gene or an expression construct (such as an expression vector) containing the coding gene into the plant.

[0022] In one or more embodiments, enhancing CRY1 expression or promoting CRY1 activation of LURP1 includes introducing the CRY1 coding gene or an expression construct (such as an expression vector) containing the coding gene into the plant.

[0023] In one or more embodiments, while upregulating the blue light-based light control system in the plant, the method further includes: increasing the intensity of blue light and increasing the duration of blue light exposure.

[0024] In another aspect of the invention, the application of a blue light-based light control system or an upregulator of the light control system in enhancing plant immunity is provided, the light control system comprising: CRY1 and its interacting protein LURP1; wherein CRY1 activates LURP1 under blue light, and LURP1 is localized to the cell membrane to enhance plant immunity.

[0025] In one or more embodiments, the light control system further includes a receptor kinase FLS2 to form the CRY1-LURP1-FLS2 pathway; preferably, FLS2 and BAK1 form an FLS2-BAK1 immune complex (i.e., form the CRY1-LURP1-FLS2-BAK1 pathway), and LURP1 promotes the formation of immune complexes and specific membrane localization of FLS2 and BAK1, enhances the activity of FLS2-BAK1, activates downstream molecules, and enhances the plant's immunity.

[0026] In one or more embodiments, the upregulator of the photocontrolled system includes: a reagent that increases LURP1 expression, a reagent that increases CRY1 expression, a reagent that promotes CRY1 activation of LURP1, a reagent that promotes N-terminal palmitoylation of LURP1, a reagent that promotes the localization of LURP1 to the cell membrane, or a reagent that promotes the interaction between LURP1 and FLS2.

[0027] In one or more embodiments, the reagent for enhancing LURP1 expression includes: a recombinant (exogenous) LURP1 encoding gene or an expression construct (such as an expression vector) containing the encoding gene.

[0028] In one or more embodiments, the reagents for enhancing CRY1 expression or promoting CRY1 activation of LURP1 include: a recombinant (exogenous) CRY1 encoding gene or an expression construct (such as an expression vector) containing the encoding gene.

[0029] In one or more embodiments, the terms "upregulation," "promotion," "enhancement," or "boost" indicate a significant upregulation, promotion, enhancement, or boost, such as an upregulation, promotion, enhancement, or boost of 5%, 10%, 15%, 20%, 40%, 60%, 80%, 90%, or higher.

[0030] In another aspect of the invention, there is provided the use of a blue light-based light control system or molecules therein in plants as molecular markers for identifying the immune capacity of plants; said light control system includes CRY1 and its interacting protein LURP1; preferably, said light control system further includes receptor-like kinase FLS2, forming a CRY1-LURP1-FLS2 pathway; more preferably, said light control system further includes BAK1, wherein FLS2 and BAK1 form an FLS2-BAK1 immune complex (i.e., forming a CRY1-LURP1-FLS2-BAK1 pathway).

[0031] In another aspect of the invention, a method is provided for identifying plant immunity or selectively screening plants with high immunity, comprising: analyzing the operation of a blue light-based light control system in the plant; said light control system includes CRY1 and its interacting protein LURP1; preferably, said light control system further includes receptor-like kinase FLS2, forming a CRY1-LURP1-FLS2 pathway; more preferably, said light control system further includes BAK1, wherein FLS2 and BAK1 form an FLS2-BAK1 immune complex (i.e., forming a CRY1-LURP1-FLS2-BAK1 pathway).

[0032] In one or more embodiments, if the CRY1 expression or activity is high, the LURP1 expression or activity is high, the N-terminal palmitoylation of LURP1 is high, the amount of LURP1 located on the cell membrane is high, and the interaction between LURP1 and FLS2 is strong, then the plant is a plant with high immunity.

[0033] In one or more embodiments, the high expression or activity of CRY1, the high expression or activity of LURP1, the high degree of N-terminal palmitoylation of LURP1, the high amount of LURP1 localized to the cell membrane, and the strong interaction between LURP1 and FLS2 are statistically or significantly increased or enhanced, such as by 5%, 10%, 15%, 20%, 40%, 60%, 80%, 90%, or even higher.

[0034] In another aspect of the present invention, a method for screening substances (including potential substances) that enhance plant immunity is provided, comprising:

[0035] (1) Under blue light, the candidate substance is added to a system with a blue light-based light control system; the light control system includes CRY1 and its interacting protein LURP1; preferably, the light control system also includes receptor kinase FLS2 to form the CRY1-LURP1-FLS2 pathway; more preferably, the light control system also includes BAK1, and the FLS2 and BAK1 form the FLS2-BAK1 immune complex (i.e., the CRY1-LURP1-FLS2-BAK1 pathway is formed);

[0036] (2) Test the system and observe the operation of the light control system. If the expression or activity of CRY1 is increased, the expression or activity of LURP1 is increased, the degree of N-terminal palmitoylation of LURP1 is increased, the amount of LURP1 located in the cell membrane is increased, and the interaction between LURP1 and FLS2 is enhanced, it indicates that the candidate substance is a substance that can be used to enhance the plant's immune ability.

[0037] In one or more embodiments, the increase in CRY1 expression or activity, the increase in LURP1 expression or activity, the increase in the degree of N-terminal palmitoylation of LURP1, the increase in the amount of LURP1 localized to the cell membrane, and the enhancement of the interaction between LURP1 and FLS2 are statistically or significantly increased or enhanced, such as by 5%, 10%, 15%, 20%, 40%, 60%, 80%, 90%, or higher.

[0038] In one or more embodiments, a control group is also included to clearly distinguish the differences between the test group and the control group in terms of CRY1 expression or activity, LURP1 expression or activity, the degree of N-terminal palmitoylation of LURP1, the amount of LURP1 localized to the cell membrane, and the interaction between LURP1 and FLS2.

[0039] In one or more embodiments, the candidate substances include (but are not limited to): regulatory molecules (such as upregulators, downregulators, mutagens, small molecule compounds (such as hormones), interference molecules, nucleic acid inhibitors, binding molecules, gene editing constructs, etc.) designed for CRY1, LURP1, FLS2 or pathways containing them such as the CRY1-LURP1-FLS2 pathway.

[0040] In one or more embodiments, the system is selected from: cell system (cell culture system), subcellular system, solution system, plant tissue system, and plant organ system.

[0041] In another preferred embodiment, the method further includes conducting further cell experiments and / or transgenic experiments on the obtained potential substances to further identify substances from the candidate substances that have an effect on regulating and enhancing immune capacity.

[0042] In another aspect of the invention, an isolated plant cell, tissue, or organ is provided, wherein recombinant expression is based on a blue light-controlled system or an upregulator of said light-controlled system; said light-controlled system includes: CRY1 and its interacting protein LURP1; preferably, said light-controlled system further includes receptor-like kinase FLS2, forming a CRY1-LURP1-FLS2 pathway; more preferably, said light-controlled system further includes BAK1, wherein said FLS2 and BAK1 form an FLS2-BAK1 immune complex; in said light-controlled system, said LURP1 is recombinantly expressed (including high expression, for example, recombinant expression in an artificially constructed expression construct), or said CRY11 is recombinantly expressed (including high expression), or said FLS2 is recombinantly expressed (including high expression).

[0043] In one or more embodiments, the recombinant expression includes: artificially created, or artificially modified.

[0044] In one or more embodiments, the recombinant expression includes introducing a foreign or heterologous gene into the cells, tissues, or organs of a plant.

[0045] In one or more embodiments, the plant cells, tissues, or organs do not directly generate living plants or serve as plant propagation material.

[0046] In one or more embodiments, the plant includes plants selected from the group consisting of: plants containing a blue light-based light control system; the light control system including CRY1 and its interacting protein LURP1; preferably, the light control system further includes a receptor-like kinase FLS2, forming a CRY1-LURP1-FLS2 pathway.

[0047] In one or more embodiments, the plant includes: a monocotyledonous plant or a dicotyledonous plant.

[0048] In one or more embodiments, the plant includes (but is not limited to): cruciferous plants, such as, but not limited to: *Mucor*, *Brassica*, *Radix*, *Isatis*, *Gnaphalium*, *Salix*, *Salix*, *Fragrance*, *Violet*, *Gnaphalium*, *Capsella*, *Nymphoides*, *Ipomoea*, etc.

[0049] In one or more embodiments, the cruciferous plants include (but are not limited to): Arabidopsis thaliana, Chinese cabbage, kale, mustard greens, radish, rapeseed, etc.

[0050] In one or more embodiments, the LURP1 comprises a protein selected from the group consisting of: (a1) a protein having the amino acid sequence of SEQ ID NO:1; (b1) a protein derived from (a1) having the protein function of (a1) formed by substituting, deleting, or adding 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 the protein function of (a1) having 80% or more (preferably 85% or more; more preferably 90% or more; more preferably 95% or more, such as 98%, 99%) homology to the protein sequence defined by (a1); or (d1) an active fragment of the protein defined by (a1), or a protein formed by adding a tag sequence, an enzyme digestion sequence, or a reporter protein to both ends thereof.

[0051] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0052] Figure 1 Blue light activates the disease resistance of plants.

[0053] ab. Blue light treatment enhanced the resistance of Arabidopsis thaliana to PstDC3000. Arabidopsis thaliana grown under short-day conditions for 4 weeks was inoculated with a bacterial suspension with an OD600 of 0.002. After inoculation, the bacteria were placed in the dark and exposed to 5 or 40 μmol·m⁻²·m⁻²·g⁻¹ ... -2 ·s -1 Short-day blue light exposure. Disease incidence was assessed 3 days post-vaccination. Error bars represent the standard deviation of six biological replicates. Different letters indicate statistically significant differences determined by Tukey's multiple comparison test (P < 0.05).

[0054] After treatment with cd. flg22, blue light caused an increase in ROS. After treatment with 100 nM flg22, in darkness (c) or at 40 μmol·m⁻¹, [the following results were observed]: -2 ·s -1 ROS levels of Col-0 and cry1 (n=12) were detected under blue light (d) conditions. Error bars represent the standard deviation of twelve biological replicates. ** indicates statistically significant differences as determined by a two-tailed Student's T test (**P<0.01).

[0055] e. Enhancement of flg22-induced MAPK phosphorylation reaction by blue light. In the dark or at 40 μmol·m -2 ·s -1 Col-0 and cry1 plants were treated with 100 nM flg22 under blue light. Their protein levels were detected using anti-pMAPK and anti-MAPK antibodies, respectively.

[0056] f. GO enrichment map of blue light-responsive genes (blue) and CRY1-dependent blue light-responsive genes (red).

[0057] Figure 2 LURP1 is involved in blue light-mediated plant immune enhancement.

[0058] a. Changes in LURP1 expression levels after blue light treatment. Ten-day-old Col-0 and cry1 seedlings were treated in the dark for 24 hours, then transferred to 40 μmol·m⁻¹ blue light. -2 ·s -1 Blue light exposure for 0.5, 1, 1.5, and 2 hours. The expression level of Col-0 in darkness was defined as "1". Error bars represent the standard deviation of the three biological replicas. Different letters indicate statistically significant differences determined by Tukey's multiple comparison test (P < 0.05).

[0059] b. Relative expression of PR1 in response to SA under dark, dark-to-blue light, or blue light conditions. Ten-day-old Col-0 seedlings were transferred to darkness or continuous blue light (40 μmol·m⁻²·s⁻¹) for 24 hours, followed by the addition of 50 μm SA (+SA) or no addition (-SA). A portion of the dark-preserved seedlings were then transferred to 40 μmol·m⁻²·s⁻¹. -2 ·s -1 Seedlings were exposed to blue light for 2 hours (D to B), and some were treated with or without SA in the dark for 2 hours (Dark). Seedlings kept under continuous blue light were also treated with or without SA for 2 hours (Blue). The expression level of Col-0 in the dark was defined as "1". Error bars represent the standard deviation of the three biological replicas. An asterisk indicates a significant difference compared to no SA according to a two-tailed Student's t-test (**P<0.01, ***P<0.001).

[0060] c. Western blotting of proteins to show changes in LURP1 protein levels in response to blue light. Ten-day-old LURP1-MYC seedlings were subjected to 24 hours of darkness and then transferred to a 40 μmol·m30 immunoblotting system. -2 ·s -1 Treat the sample with blue light (top) or in blue light for 24 hours, then transfer to darkness (bottom) for 0.5, 1, 1.5, or 2 hours. Protein levels in the sample are detected using anti-MYC antibody, with Ponceau S staining used as a loading control.

[0061] d. In lurp1 mutant plants, the enhancing effect of blue light on pathogen resistance was weakened. This was observed at light intensities of 5 or 40 μmol·m⁻¹. -2 ·s -1The number of PstDC3000 was determined in plants (Col-0, lurp1-3, and LURP1-MYC) grown under specific conditions. Adult Arabidopsis plants were inoculated with PstDC3000 (OD600 = 0.002), and bacterial counts were determined 3 days post-inoculation. Error bars represent the standard deviation of three six-biological replicates. Different letters indicate statistically significant differences (P < 0.05) as determined by Tukey's multiple comparison test.

[0062] ef.flg22 in darkness or blue light (40 μmol·m -2 ·s -1 ROS bursts were induced in Arabidopsis thaliana under [treatment name missing]. Error bars represent the standard deviations of twelve biological replicates (n=12). Different letters indicate statistically significant differences (P<0.05) as determined by Tukey's multiple comparison test.

[0063] RT-qPCR analysis of FRK1 transcriptional levels 1 hour after g. flg22 treatment. FRK1 expression levels after ddH2O treatment were defined as "1". At5g15400, encoding a U-box domain protein, was used as an internal control. Error bars represent the standard deviation of three biological replicates. Different letters indicate statistically significant differences determined by Tukey's multiple comparison test (P < 0.05).

[0064] h. Col-0, lurp1-3 and LURP1-MYC seedlings grown for 10 days were treated with 100 nM flg22 in the dark or under 40 μmol·m-2·s-1 blue light, and the phosphorylation of MAPK was detected by anti-pMAPK and anti-MAPK antibodies, respectively.

[0065] ij. The number of PstDC3000 bacteria in Col-0, lurp1-1, CRY1-FLAG and CRY1-FLAG / lurp1-1 plants (i) or Col-0, LURP1-GFP, LURP1-GFP / cry1 plants (j) 3 days after inoculation.

[0066] Figure 3 Flg22 induces LURP1 cell membrane localization via N-terminal palmitoylation.

[0067] a.LURP1-mCherry and LURP1 C13A Subcellular localization of -mCherry before and after flg22 treatment (1 μM) for 5 min. Cell membrane labeled with Lti6b-GFP. Scale bar indicates 40 μm.

[0068] b. Immunoblotting showed the plasma membrane localization of LURP1-GFP protein in 35S::LURP1-GFP seedlings grown for 10 days before and after treatment with 1 μM flg22.

[0069] c. flg22-induced palmitoylation of LURP1. The palmitoylation level of LURP1 protein after treatment with 1 μM flg22 in 10-day-old 35S::LURP1-FLAG seedlings was determined by a method dependent on the selective cleavage of the thioester bond between the acyl and cysteine ​​residues by neutral hydroxylamine. Untreated samples were used as controls. LURP1 protein was detected using an anti-FLAG antibody.

[0070] d. Treat 35S::LURP1-GFP and 35S::LURP1-GFP grown for 10 days under blue light with 1 μM flg22. C5A -GFP and 35S::LURP1 C13A -GFP palmitoylation level in Arabidopsis thaliana seedlings.

[0071] e. Three days after inoculation under blue light, in Col-0, lurp1-3, LURP1 C13A - The content of PstDC3000 in GFP / lurp1-3 plants.

[0072] Figure 4 LURP1 interacts with FLS2 and enhances its immune activity.

[0073] a. BiFC experiments showed that LURP1 interacts with FLS2 at the cell membrane. LURP1-nYFP or LURP1 C13A -nYFP and FLS2-cYFP were co-expressed in *Nicotiana benthamiana*. Yellow fluorescence of *Agrobacterium* was observed 48 hours after infection using confocal microscopy. Scale bar indicates 100 μm.

[0074] b. Immunoprecipitation of LURP1 and FLS2 in vivo. Seedlings grown for 10 days were transferred to liquid medium and incubated overnight, then treated with (1 μM) flg22 for 5 minutes. Total protein was extracted from Col-0 or LURP1-FLAG seedlings, enriched with anti-FLAG antibody, and the co-precipitation of FLS2 was detected using anti-FLS2 antibody.

[0075] c. In lurp1-3, flg22-induced interaction between FLS2 and BAK1 is weakened. FLS2-GFP was enriched using an anti-GFP antibody, and BAK1 co-precipitation was detected using an anti-BAK1 antibody. * indicates a non-specific band.

[0076] d. In lurp1-3, Flg22-induced BIK1 phosphorylation was reduced. BIK1-HA in wild-type, cry1, or lurp1-3 backgrounds was reduced in the dark and at 40 μmol·m -2 ·s -1 Proteins were extracted after treatment with flg22 (1 μM) under blue light, and phosphorylated and non-phosphorylated forms of BIK1-HA were detected using an anti-HA antibody.

[0077] e. Predicting the structure of LURP1 using AlphaFold.

[0078] f. Interaction model: Blue light regulates PTI signaling through its receptor CRY1. TF: Transcriptional activation. Detailed Implementation

[0079] To explore the molecular mechanisms by which light regulates plant disease resistance, the inventors conducted extensive research and discovered a gene, LURP1, that is simultaneously induced by blue light and pathogens, and that positively regulates plant immunity under blue light. LURP1, along with upstream and downstream molecules (including CRY1 and FLS2), constitutes a novel blue light-based light-controlled system. Upon pathogen invasion, LURP1 can be palmitoylated onto the cell membrane and activate the important receptor kinase FLS2 in the PTI (phosphorus kinase inhibitor). The technical solution of this invention contributes to molecular design breeding, providing a theoretical basis for cultivating high-quality varieties that can resist pathogen invasion while also ensuring high yield.

[0080] As used herein, the term "plant (crop)" refers to a plant containing the aforementioned blue light-based light control system; preferably containing CRY1 and its interacting protein LURP1 or homologs thereof, and more preferably also containing FLS2 and BAK1 or homologs thereof. The plant includes monocotyledonous or dicotyledonous plants. For example, the plant may include plants of the Brassicaceae, Solanaceae, Poaceae, Euphorbiaceae, etc. More preferably, the plant is a Brassicaceae plant. More preferably, the plant is a crop (such as a cash crop). For example, the "plant" includes, but is not limited to, Arabidopsis thaliana, Chinese cabbage, kale, mustard greens, radish, rapeseed, etc.

[0081] Regarding "control plants," selecting appropriate control plants is a routine part of experimental design. These can include corresponding wild-type plants or transgenic plants without the target gene. Control plants are generally the same plant species or even varieties of the same species or class as the plant being evaluated. Control plants can also be individuals from transgenic plants that have lost their transgenic components due to segregation. As used in this article, control plants refer not only to whole plants but also to plant parts, including seeds and seed portions.

[0082] It should be understood that, with the guidance of the technical solutions of this invention, those skilled in the art will readily conceive of changing various plant species to achieve the same or similar technical effects, and these variations are also included in this invention.

[0083] As used in this invention, the term "(signaling) pathway" refers to a signaling system formed by the mutual constraints or interactions of a series of proteins or genes, which generally leads to the occurrence of certain cellular events.

[0084] Blue light-based light control system

[0085] In this invention, the blue light-based light control system includes: the CRY1 gene (and / or its encoded protein) and the LURP1 gene (and / or its encoded protein); preferably, it also includes: the FLS2 gene (and / or its encoded protein) and the BAK1 gene (and / or its encoded protein).

[0086] As used in this invention, the terms “CRY1-LURP1-FLS2 pathway” and “CRY1 / LURP1 / FLS2 pathway” are used interchangeably.

[0087] As used herein, the molecules in the blue light-based light control system include genes or proteins from Arabidopsis thaliana (At), genes or proteins that are homologous to genes or proteins from Arabidopsis thaliana, contain substantially the same structural domains, and have substantially the same functions.

[0088] As used herein, the protein amino acid sequence of “LURP1”, for example as shown in SEQ ID NO:1, may also be its homolog (homologous protein or homologous gene).

[0089] As used herein, the protein amino acid sequence of “CRY1”, for example as shown in GenBank accession number 826470, may also be its homologs (homologous proteins or homologous genes).

[0090] As used herein, the protein amino acid sequence of “FLS2”, for example as shown in GenBank accession number 834676, may also be its homologs (homologous proteins or homologous genes).

[0091] As used herein, the protein amino acid sequence of “BAK1”, for example as shown in GenBank accession number 829480, may also be its homologs (homologous proteins or homologous genes).

[0092] Those skilled in the art will recognize 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 / ).

[0093] In this invention, the molecules (proteins) in the light-controlled system also include their fragments, derivatives, and analogs. As used herein, the terms "fragment," "derivative," and "analyte" refer to protein fragments that substantially retain the same biological function or activity as the protein, and may be (i) proteins with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) proteins having substituent groups in one or more amino acid residues; or (iii) proteins formed by fusing additional amino acid sequences to the protein sequence; or (iv) proteins with one or more amino acid residues (or domains) removed (or truncated), for example, by removing inactive sequence regions. These fragments, derivatives, and analogs are within the scope known to those skilled in the art as defined herein. All bioactive fragments of the molecules in the light-controlled system can be used in this invention.

[0094] As used herein, "variant," "mutant," "mutant protein," "mutant form," "Mt," or "Mt" refers to a peptide or protein whose amino acid sequence has been altered compared to a reference sequence (wild-type sequence) through the insertion, deletion, or substitution of one or more amino acids, but which retains at least one biological activity; at the nucleic acid level, the corresponding coding site has changed compared to the corresponding reference sequence. In this invention, "mutation" generally refers to a mutation closely related to the occurrence of disease. The "inserted or substituted amino acid" can be a natural amino acid or a non-natural amino acid.

[0095] It should be understood that while the molecules (genes or proteins) in the light control system of the present invention are preferably obtained from cruciferous plants, particularly Arabidopsis thaliana, genes (such as genes with degeneracy) from other plants that are highly homologous to the corresponding genes or proteins in Arabidopsis thaliana (e.g., having more than 80%, such as 85%, 90%, 95%, or even 98% sequence identity) 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.

[0096] Vectors containing the said coding sequence, and host cells genetically engineered using the said vector or protein-coding sequence, are also included in this invention. Methods well known to those skilled in the art can be used to construct suitable expression vectors.

[0097] The host cell is usually a plant cell. Plant transformation can be achieved using methods such as Agrobacterium-mediated transformation or gene gun transformation, with Agrobacterium-mediated transformation being preferred. Transformed plant cells, tissues, or organs can be regenerated into plants using conventional methods, thereby obtaining plants with altered traits compared to the wild type.

[0098] When used as targets for artificial regulation or in the creation of screening systems, the proteins or encoding genes mentioned above can be naturally occurring, such as those purified and isolated from mammals or plants; or they can be recombinantly prepared, for example, recombinant proteins can be produced using conventional gene recombination techniques. Furthermore, any variations that do not affect the biological activity of these proteins are acceptable, such as derivatives or variants whose function remains unchanged.

[0099] Applications of blue light-based light control systems

[0100] Through extensive screening and meticulous analysis, the inventors have revealed a blue light control system containing LURP1 and its application in activating plant immunity.

[0101] The inventors discovered that blue light can enhance plant immune responses and resistance to pathogens. LURP1, transcriptionally activated by CRY1, participates in blue light-mediated plant immune enhancement. Furthermore, N-terminal palmitoylation of LURP1 is triggered, leading to cell membrane localization of the LURP1 protein. Cell membrane-localized LURP1 enhances plant immunity under blue light. Further research suggests that LURP1 positively regulates immune signaling by enhancing the formation and function of the FLS2-BAK1 receptor complex.

[0102] Therefore, this invention reveals that LURP1 is activated by the blue light receptor CRY1 under blue light. After pathogen infection, the LURP1 protein undergoes N-terminal palmitoylation, localizing to the cell membrane to bind to the receptor FLS2, enhancing its immune activation activity, thereby strengthening the plant's disease resistance.

[0103] Based on the aforementioned new discoveries of the inventors, the use of the light control system, or molecules in the system, or their regulators (upregulators), is provided for: enhancing the immunity of plants, including: enhancing the disease resistance of plants (the ability to resist pathogens). The disease resistance includes: the ability to resist pathogens. The disease resistance described in this invention refers to the ability to resist plant diseases compared to a corresponding wild-type control.

[0104] The ability of light to regulate plant disease resistance can be utilized by increasing light intensity, duration, and altering the light spectrum to enhance plant immunity.

[0105] Protein pesticides that activate plant immune responses can also be designed based on the characteristics of the light control system or the molecules in the system described in this invention, which can help plants resist pathogens in a more environmentally friendly and safer way, reduce dependence on chemical pesticides, and provide safer food.

[0106] In this invention, the pathogen may include: a pathogen that induces / triggers activation of the blue light-based light control system or molecules within the system after infecting the plant. The pathogen includes plant pathogens (such as plant pathogenic bacteria), where "plant pathogen" refers to any pathogen that causes a disease state in a plant, including bacteria, fungi, viruses, viroids, insects, nematodes, mycoplasma, etc. Resistance or "disease resistance" refers to a reduction or weakening of disease symptoms in the plant after pathogen infection. 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 the crop. Disease symptoms caused by pathogens include the size and / or frequency of necrotic or yellowish lesions on plant tissues, brown lesions (often with a pale brown to slightly white center, accompanied by a dark, slightly purple to black outer edge, gradually surrounded by a yellowish-brown band); the size and / or frequency of canker, black spot (also known as dark leaf spot), downy mildew, etc.

[0107] In this invention, the upregulators of the light-controlled system or the molecules within it include promoters, agonists, activators, etc. The terms "upregulation" and "promotion" include "upregulation" and "promotion" of protein activity or protein expression, and these are statistically significant "upregulation" and "promotion." Any substance that can increase the activity of the light-controlled system or the molecules within it (including upstream and downstream proteins), improve the stability of the light-controlled system or the molecules within it, upregulate the expression of the light-controlled system or the molecules within it, increase the effective duration of action of the light-controlled system or the molecules within it, or increase the activation level of individual molecules can be used in this invention as substances useful for upregulating the light-controlled system or the molecules within it. These substances can be compounds, small chemical molecules, or biomolecules. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level.

[0108] In a preferred embodiment of the present invention, the upregulator may include: a reagent that increases LURP1 expression, a reagent that increases CRY1 expression, a reagent that promotes CRY1 activation of LURP1, a reagent that promotes N-terminal palmitoylation of LURP1, a reagent that promotes the localization of LURP1 to the cell membrane, or a reagent that promotes the interaction between LURP1 and FLS2.

[0109] It should be understood that, after understanding the function of the blue light control system containing LURP1 (preferably, also including its upstream and downstream genes), various methods well known to those skilled in the art can be used to regulate the expression or activity of the light control system or its molecules, or to regulate its upstream or downstream genes. For example, various methods well known to those skilled in the art can be used for recombinant expression or overexpression. These methods are all included in this invention.

[0110] The light control system of the present invention can also be used to screen substances (including potential substances) that enhance plant immunity. The screening method includes: (1) adding the candidate substance to a system with a blue light-based light control system; the light control system includes CRY1 and its interacting protein LURP1; preferably, the light control system also includes receptor kinase FLS2 to form a CRY1-LURP1-FLS2 pathway; more preferably, the light control system also includes BAK1, and the FLS2 and BAK1 form an FLS2-BAK1 immune complex (i.e., forming a CRY1-LURP1-FLS2-BAK1 pathway); (2) detecting the system and observing the operation of the light control system therein. If the expression or activity of CRY1 is increased, the expression or activity of LURP1 is increased, the degree of N-terminal palmitoylation of LURP1 is increased, the amount of LURP1 located on the cell membrane is increased, and the interaction between LURP1 and FLS2 is enhanced, then the candidate substance is a substance that can be used to enhance plant immunity.

[0111] 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 all be used in this invention. The candidate substances can be selected from: peptides, polymeric peptides, peptide-like substances, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, and nucleic acid sequences, etc. Those skilled in the art understand how to select appropriate screening methods based on the type of substance to be screened. Through large-scale screening, a class of potential substances that specifically regulate the light control system and thus have a regulatory effect on plant immunity can be obtained.

[0112] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Science Press, or according to the manufacturer's recommendations.

[0113] Materials and methods

[0114] 1. Plant materials

[0115] The wild-type Arabidopsis thaliana material used in this invention is Col-0 (Columbia), and the overexpression and mutant materials constructed are all based on Col-0.

[0116] The tobacco material used in this invention is Nicotiana benthamiana.

[0117] The mutant material lurp1-1 (SALK_039201C) used in this invention is a T-DNA insertion mutant ordered from ABRC (Arabidopsis Germplasm Resource Center, USA). The Lurp1-3 mutant was constructed using CRISPR-Cas9 technology.

[0118] Overexpression material is the LURP1 coding sequence or LURP1 C13A (The amino acid sequence of LURP1 is mutated from C to A at position 13), LURP1 C5A The coding sequence (LURP1 amino acid sequence 5 is mutated from C to A) was cloned into pEAGD (LURP1-MYC), pCambia2300 (LURP1-GFP), or pCambia1300 (LURP1-FLAG) and transformed into Arabidopsis thaliana.

[0119] fls2 mutant and BIK1-HA were obtained from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.

[0120] Obtained by double mutation or double overexpression genetic hybridization.

[0121] 2. Bacterial infection and growth

[0122] Arabidopsis plants were grown under 12 hours of white light and 12 hours of darkness for 4–5 weeks. *Pseudomonas syringae* PstDC3000 was cultured at 28°C for 5–8 hours in Luria Marine (LM) medium containing 50 mg / L rifampicin, with a cell density of OD600 = 0.8–1.0. Bacterial cells were collected and resuspended in sterile water with OD600 = 0.002. The bacterial suspension was infiltrated into the leaves using a 1 mL needle-free syringe. The plants were covered with transparent plastic domes to maintain high humidity. After three days, three leaf discs were removed from three different leaves using a 7 mm diameter perforator as a biological replicate, and six biological replicates were taken from each treatment. The leaf discs were ground in sterile water and incubated in LM solid medium containing rifampicin at 28°C for 36 hours for colony counting.

[0123] 3. Flg22-induced reactive oxygen species burst

[0124] The 22 conserved amino acids at the N-terminus of bacterial flagellin are called Flag22 (Flagellin 22). Arabidopsis thaliana was grown for 4–5 weeks under 12 hours of white light and 12 hours of darkness. The day before ROS measurement, leaf discs were obtained using a perforator (5.5 mm diameter) and floated in 96-well plates with 200 μL ddH2O. Before measurement, water was removed, and ROS detection solution (30 mg / L luminol (Sigma-Aldrich), 20 mg / L horseradish peroxidase (Sigma-Aldrich)) and 100 nM Flag22 were added. TriStar was used. 2 The LB 942 multimode microplate reader (BERTHOLD TECHNOLOGIES) monitored luminescence for 45 minutes.

[0125] 4. Bi-fluorescent molecular complementarity assay (BiFC assay)

[0126] LURP1 and FLS2 / CRY1 were constructed on vectors containing nYFP and cYFP, respectively, and transformed into Agrobacterium. After identification, transient expression transformation experiments were performed on tobacco, and the results were observed using a fluorescence microscope.

[0127] 5. Pull-down experiment

[0128] Add the recombinant protein to be tested and the corresponding control protein sample to a buffer (20mM Tris (pH 7.4), 100mM NaCl, 12mM MgCl2, 1mM DTT) and incubate at 37℃ for 1 hour. Aliquot 15-20 μL of GST beads into each tube and wash twice with 2×extraction buffer [100mM Tris·HCl (pH 7.5), 300mM NaCl, 2mM EDTA (pH 8.0), 1% Trion X-100, 10% (vol / vol) glycerol, protease inhibitor mixture (AMRESCO)]. Add 1 mL of 2×extraction buffer. Add the reacted protein to the washed beads and incubate at 4℃ on a silent mixer for 2-3 hours. Centrifuge at 2000 rpm for 2 min and discard the supernatant. Wash the beads 8 times with 2×extraction buffer, 2000 rpm for 2 min each time, and discard the supernatant. Aspirate the water layer from the beads, add approximately 50 μL of 1×SDS-PAGE loading buffer (250 mM Tris-HCl, pH 6.8, 10% SDS, 1% bromophenol blue, 50% glycerol, 500 mM DTT), and boil at 95°C for 10 min. After centrifugation, collect the supernatant for Western blot analysis.

[0129] 6. Protein co-precipitation assay (co-IP)

[0130] Grind the plant material into powder using liquid nitrogen, add an equal volume of 2×Extraction buffer, and incubate for 1-2 hours on a silent mixer at 4°C. Centrifuge at 12000 rpm for 10 min at 4°C and discard the precipitate. Filter the supernatant through a membrane to thoroughly remove plant residue. Aliquot 15-20 μL of the FLAG beads into each tube and wash twice with 2×Extraction buffer. Add the prepared plant extract to the washed FLAG beads and incubate for 3-4 hours on a silent mixer at 4°C. Centrifuge at 2000 rpm for 2 min and discard the supernatant. Wash the beads 8 times with 2×Extraction buffer, 2000 rpm for 2 min each time, and discard the supernatant. Aspirate the water layer from the beads, add approximately 50 μL of 1×SDS-PAGE loading buffer, and boil at 95°C for 10 min. After centrifugation, collect the supernatant for Western blot analysis.

[0131] 7. Real-time quantitative polymerase chain reaction (qPCR)

[0132] Using cDNA as a template, qPCR detection was performed using Takara's qPCR reaction reagents and system. The prepared mixture was placed in an MX3000 (Stratagene) system for execution. The execution program was: 95℃, 30s per cycle; 95℃, 5s, 60℃, 30s, 40-50 cycles; ACT7 was used as an internal control, and each experiment had two technical replicates and at least two biological replicates.

[0133] 8. S-palmitoylation assay

[0134] S-palmitoylation of LURP1 was determined using a modified acyl RAC method. Total protein was extracted using a buffer containing 50 mM N-ethylmaleimide (Sigma, 04260) (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 5% glycerol, 1% Triton X-100, 1% SDS, 1x protease inhibitor). After overnight incubation with shaking at 4°C, 3 volumes of acetone were added to precipitate the protein. The precipitate was washed three times and redissolved in 150 μL of 2% SDS buffer (50 mM Tris, 5 mM EDTA, pH 7.4). 10 μL of the supernatant was used as input, and the remainder was divided in half and incubated with or without 0.7 M hydroxylamine (Sigma). The "+HA" buffer consisted of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 5% glycerol, 0.2% Triton X-100, 1x protease inhibitor, and 0.7 M hydroxylamine. The "-HA" buffer had the same composition, except that it lacked hydroxylamine. 20 μL of activated thiol Sepharose beads (WSAC, CS-A32-01, Beijing) were added to both the "+HA" and "-HA" samples and incubated for 3 hours. The beads were washed four times, then boiled at 60 °C for 10 minutes before Western blotting.

[0135] 9. Sequence Information

[0136] The amino acid sequence of LURP1 (SEQ ID NO:1):

[0137] MQQPCVIVGSKYCSPNPVGLAIVRKVMKITDGNFVITSADGKLLFKVKDPLFSLHGKRILLDCSGAKVLTLRGKMMTMHDRWQVFRGGSTEEGALLYTVKRSS MIQLAPKLEVFLANNVEEKICDFKVKGAWLDDSCVVYAGDSDTIIAHMCGKQTMRGFFFGKDHFSVTVDKNVDYAFIASLIVILVEIEKAGFITKMTTQMIIGF

[0138] Example 1: Blue light can enhance the immune response and resistance to pathogens in plants.

[0139] The inventors investigated the effect of blue light treatment on pathogen infection. *Pseudomonas syringae* PstDC3000 was inoculated into *Arabidopsis thaliana*. Compared with dark treatment, treatment with a low dose of blue light (Blue5; 5 μmol·m⁻¹) significantly improved the bacterial infection rate. -2 ·s-1 Compared to this, using a higher blue light intensity (Blue40; 40 μmol·m⁻¹) -2 ·s -1 Plants treated with this method showed significantly enhanced resistance to PstDC3000. Figure 1 ab) indicates that blue light induces immune enhancement in a dose-dependent manner. The blue light signal is sensed by the receptor cryptochrome (CRY).

[0140] The inventors investigated whether the blue light-mediated increase in resistance depended on CRY1. Although the bacterial population in the cry1 mutant plants still showed a slight decrease after blue light treatment, the enhanced resistance level was significantly lower compared to the wild-type plants, indicating that blue light enhances plant disease resistance through photoreceptor sensing.

[0141] Immune signals can be activated upon PAMP recognition. A 22-amino acid peptide derived from bacterial flagellin is a PAMP called flg22 (which mimics the immune signaling activation caused by pathogen infection). Flg22 treatment leads to a rapid burst of reactive oxygen species (ROS), a hallmark of the early PAMP response. ROS detection showed that wild-type plants exhibited a significantly increased ROS burst under blue light compared to the cry1 mutant. Figure 1 c). Conversely, WT and cry1 plants exhibited similar ROS burst levels in the dark ( Figure 1 d). Phosphorylation of flg22-triggered mitogen-activated protein kinases (MAPKs) was also increased. Figure 1 e). The blue light-enhanced response in the cry1 mutant was eliminated or almost eliminated. Figure 1 e).

[0142] These results support the direct regulation of pattern-triggered immunity (PTI) by blue light sensing.

[0143] Example 2: LURP1 participates in blue light-mediated plant immune enhancement

[0144] To elucidate the molecular mechanism of blue light-mediated flg22-triggered enhanced immune signaling, the inventors determined transcriptomic changes in the cry1 mutant compared to wild-type Col-0. Ten-day-old seedlings grown under short-day conditions were moved to darkness for 24 hours, followed by 1 hour of blue light treatment. Changes were observed in 3359 genes, including 2243 genes regulated in a CRY1-dependent manner. Blue light-responsive genes are primarily involved in light signal transduction, oxygen response, photosynthesis, and circadian rhythms. Figure 1 Interestingly, CRY1-dependent blue light-responsive genes are primarily involved in stress responses, including immune responses and responses to the defensive plant hormones SA and jasmonic acid (JA). Figure 1f). Comparison of the inventors' transcriptome data with previously published transcriptome data treated with flg22 or PstDC3000 (GSE5615) revealed 172 co-regulated genes, 7 of which have known disease resistance functions. Through Q-PCR and phenotypic validation, the inventors discovered that LURP1 is involved in the regulation of blue light-activated disease resistance processes.

[0145] The inventors discovered that LURP1 expression is low in plants grown in the dark, but can be rapidly induced after blue light treatment. Figure 2 a). This induction disappeared in cry1 mutant plants. Observations also showed that LURP1 protein accumulation increased rapidly after blue light treatment. Figure 2 c). When plants transition from blue light to darkness, LURP1 protein levels rapidly decline. This evidence supports the view that LURP1 is a blue light-responsive protein regulated by blue light at both the transcriptional and post-translational levels.

[0146] To investigate the role of LURP1 in blue light-mediated immune signaling, the inventors constructed a mutant with a complete CDS deletion using the CRISPR-Cas9 method, named lurp1-3. When subjected to low-intensity blue light (5 μmol·m⁻¹), the LURP1 was analyzed. -2 ·s -1 When treated with ), lurp1-3 showed similar resistance to PstDC3000 as wild-type Arabidopsis; however, when treated with high intensity (40 μmol·m⁻¹), the resistance was significantly reduced. -2 ·s -1 Blue light treatment did not improve bacterial resistance in lurp1-3 to a level similar to that in wild-type plants. Figure 2 d). Furthermore, even under low levels of blue light, transgenic plants expressing 35S-LURP1-MYC (plants overexpressing LURP1 driven by the 35S promoter) showed significantly enhanced resistance to PstDC3000, with the enhanced effect being more pronounced under relatively high levels of blue light. Figure 2 d) suggests that this is due to the accumulation of higher levels of LURP1 protein than WT.

[0147] The inventors also measured the ROS surge after flg22 treatment in lurp1-3 and 35S::LURP1-MYC plants. In darkness, ROS levels increased in 35S::LURP1-MYC but remained unchanged in lurp1-3. Figure 2 e). Under blue light processing, the ROS levels in WT and 35S::LURP1-MYC are higher than those in lurp1-3 ( Figure 2f). Consistent with this observation, flg22 treatment decreased the induction of FRK1 gene expression in lurp1-3, but significantly increased 35S::LURP1-MYC. Similarly, in lurp1-3, blue light-enhanced flg22 induced reduced MAPK phosphorylation (f). Figure 2 g), and constitutive expression of lurp1 leads to an overall increase in flg22-guided MAPK phosphorylation in both dark and blue light. Figure 2 These results indicate that LURP1 plays an important role in the blue light-enhanced flg22-activated immune response.

[0148] To further elucidate the relationship between LURP1 and CRY1 in blue light-mediated immune regulation, the inventors prepared transgenic plants expressing 35S::CRY1-FLAG in both WT and lurp1-1 backgrounds (plants with 35S promoter-driven CRY1 overexpression). When the plants were treated with low-intensity blue light, overexpression of CRY1 in the wild type further increased bacterial resistance. However, this enhancement was attenuated in the lurp1-1 background. Figure 2 i) indicates that LURP1 functions downstream of CRY1, consistent with CRY1's transcriptional activation of lurp1. Furthermore, in a cry1 mutant background, LURP1-GFP overexpression no longer induced enhanced resistance (i). Figure 2 j).

[0149] These results indicate that, in addition to transcriptional activation, CRY1 also regulates LURP1 through other mechanisms, thereby modulating plant immunity.

[0150] Example 3: N-terminal palmitoylation enables LURP1 to localize to the cell membrane.

[0151] PAMP activation of immune signaling depends on its corresponding cell surface binding immune receptors. Flg22 is directly recognized by the receptor kinase FLAGELLIN SENSING 2 (FLS2), which forms a receptor complex with the co-receptor BAK1 to activate the immune response. Since the lurp1 mutant's response to flg22 is characterized by ROS bursts and FRK1-induced reduction, the inventors investigated whether LURP1-mediated enhanced defense under blue light requires FLS2. To this end, LURP1-FLAG was overexpressed in WT (Col-0) and fls2 mutant backgrounds, respectively. The results showed that in the fls2 mutant, blue light-enhanced resistance to PstDC3000 was almost eliminated.

[0152] In fact, blue light-mediated resistance enhancement was no longer observed in fls2-treated plants, indicating that fls2 plays an important role. FLS2 is primarily localized on the cell membrane. Therefore, the inventors constructed a transgenic Arabidopsis thaliana of 35S::LURP1-mCherry to detect the subcellular localization of LURP1. flg22 treatment significantly increased the cell membrane localization of LURP1. Figure 3 a). Cell membrane extraction experiments confirmed this observation. LURP1 protein levels were largely induced by blue light, rather than by flg22. However, significant accumulation of LURP1 protein on the plasma membrane was only observed under flg22 treatment and blue light. Figure 3 b). Furthermore, in the cry1 mutant, LURP1 does not localize to the cell membrane. Figure 3 b). This is consistent with previous observations that overexpression of LURP1 in the cry1 mutant does not enhance blue light-dependent immunity. Figure 2 These results indicate that, in addition to transcriptional activation, CRY1 also regulates LURP1 function at the post-translational level.

[0153] N-terminal palmitoylation can mediate protein localization to the cell membrane. The inventors' analysis showed that LURP1 has two potential palmitoylation sites: Cys5 and Cys13; and LURP1 can be palmitoylated. Importantly, palmitoylation of LURP1 was significantly enhanced after treatment with flg22. Figure 3 c). Site-directed mutagenesis of the predicted palmitoylation sites revealed that LURP1 C13A The level of palmitoylation in the sample was significantly reduced. Figure 3 d). And LURP1 C13A After treatment with flg22, it completely lost its cell membrane localization. Figure 3 bc). Furthermore, in blue light-induced immune enhancement, LURP1... C13A Mutant phenotypes that cannot complement lurp1-3 ( Figure 3 e).

[0154] These results indicate that N-terminal palmitoylation is triggered to localize the LURP1 protein to the cell membrane, and cell membrane-localized LURP1 enhances plant immunity under blue light.

[0155] Example 4: LURP1 promotes specific membrane localization of the FLS2-BAK1 immune complex, enhancing immune activity. Since LURP1-mediated resistance to PstDC3000 requires the flg22 receptor FLS2, the inventors investigated whether LURP1 could interact with FLS2. In a bimolecular fluorescence complementation experiment, LURP1 and FLS2 could interact on the cell membrane (…). Figure 4a). This interaction in LURP1 C13A The disappearance of LURP1-FLAG further confirms the importance of cell membrane localization in this interaction. Furthermore, in in vivo co-precipitation experiments, LURP1-FLAG could only interact with FLS2 after flg22 treatment. Figure 4 b). This is consistent with the previous results, that is, LURP1 can only be localized on the cell membrane and interact with FLS2 located on the cell membrane after flg22 treatment.

[0156] Can LURP1 regulate the function of FLS2 as a receptor kinase? FLS2 and its co-receptor BAK1 form a complex in the presence of flg22. However, in lurp1-3 plants, flg22-induced FLS2-BAK1 interaction is weakened. Figure 4 c) indicates that LURP1 promotes the formation of the FLS2 and BAK1 receptor complex. In the FLS2 signaling pathway, BIK1 is phosphorylated after the receptor complex is formed. The inventors found that under blue light, the phosphorylation level of BIK1 in the wild type was significantly higher than that in darkness. Figure 4 d). However, this enhancement was significantly suppressed in the cry1 and lurp1-3 mutants ( Figure 4 d) This is consistent with the blue light-triggered enhancement of immune signals in a cry1 and lurp1-dependent manner.

[0157] These results indicate that under blue light, LURP1 positively regulates immune signaling by enhancing the formation and function of the FLS2-BAK1 receptor complex.

[0158] The structural model of LURP1 was generated using AlphaFold. Figure 4 e) The study found that LURP1 is significantly similar to phospholipid crawling enzymes (PLSCRs) in mammals. Palmitoylated PLSCRs are located on the cell membrane and can regulate the activity of the receptor kinase EGFR. LURP1 shares similar domain structures with human PLSCRs, including transmembrane helices and Ca2+ binding motifs. Figure 4 e). Interestingly, cell surface-localized immune receptor complexes, including FLS2-BAK1, are located within specific plasma membrane nanostructure domains. The results also suggest that LURP1, through interaction with FLS2, facilitates the specific membrane localization of the FLS2-BAK1 immune complex, thereby enhancing its activity. Pathogen-induced palmitoylation of LURP1 is important for enhancing immune signaling.

[0159] In summary, this invention reveals that LURP1 is activated by the blue light receptor CRY1 under blue light. After pathogen infection, the LURP1 protein undergoes N-terminal palmitoylation, localizing to the cell membrane and binding to the receptor FLS2, enhancing its immune activation activity and thus strengthening the plant's disease resistance. Figure 4 f).

[0160] Example 5: Screening Method

[0161] Test group: Arabidopsis thaliana cell lines (expressing the light control system of the present invention, including CRY1, LURP1, FLS2, BAK1), and subjected to blue light irradiation, and given candidate substances;

[0162] Control group: Arabidopsis cell lines (expressing the light control system of the present invention, including CRY1, LURP1, FLS2, BAK1) were irradiated with blue light but without the candidate substance.

[0163] The operation of the light control system in the test group and the control group was tested and compared. If the expression or activity of CRY1, LURP1, N-terminal palmitoylation of LURP1, the amount of LURP1 localized to the cell membrane, and the interaction between LURP1 and FLS2 were statistically enhanced (e.g., enhanced by more than 10%) in the test group, then the candidate substance is considered a substance that can be used to enhance plant immunity.

[0164] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for enhancing plant immunity, comprising: Upregulates blue light-based light control systems in plants; these light control systems include CRY1 and its interacting protein LURP1. The CRY1 activates LURP1 under blue light, and LURP1 is located on the cell membrane, enhancing the plant's immune ability.

2. The method as described in claim 1, characterized in that, The light control system also includes receptor-like kinase FLS2, forming the CRY1-LURP1-FLS2 pathway; Preferably, CRY1 activates LURP1 under blue light. When plants are infected by pathogens, LURP1 localizes to the cell membrane, binds to receptor kinase FLS2, and enhances the immune activation activity of FLS2, thereby enhancing the plant's immunity. Preferably, when a plant is infected by a pathogen, the LURP1 undergoes N-terminal palmitoylation and is localized to the cell membrane; preferably, the cysteine ​​residue at position 13 is palmitoylated. Preferably, the light control system further includes BAK1, and FLS2 and BAK1 form an FLS2-BAK1 immune complex. LURP1 promotes the formation of the immune complex and specific membrane localization of FLS2 and BAK1, enhances the activity of FLS2-BAK1, activates downstream molecules, and enhances the plant's immunity.

3. The method as described in claim 1 or 2, characterized in that, The upregulation of blue light-based light control systems in plants includes: increasing LURP1 expression, increasing CRY1 expression, promoting CRY1 activation of LURP1, promoting N-terminal palmitoylation of LURP1, promoting LURP1 localization to the cell membrane, or promoting the interaction between LURP1 and FLS2 to enhance the immune activation activity of FLS2. Preferably, the enhancement of LURP1 expression includes: introducing the LURP1 coding gene or an expression construct containing the coding gene into the plant; Preferably, the enhancement of CRY1 expression or promotion of CRY1 activation of LURP1 includes: introducing the CRY1 encoding gene or an expression construct containing the encoding gene into the plant; Preferably, while upregulating the blue light-based light control system in the plant, the method further includes: increasing the intensity of blue light and increasing the duration of blue light exposure.

4. The application of a blue light-based light control system or an upregulator of said light control system in enhancing plant immunity, said light control system comprising: CRY1 and its interacting protein LURP1; The CRY1 activates LURP1 under blue light, and LURP1 is located on the cell membrane, enhancing the plant's immune ability.

5. The application as described in claim 4, characterized in that, The light control system also includes receptor kinase FLS2, forming the CRY1-LURP1-FLS2 pathway; preferably, FLS2 and BAK1 form an FLS2-BAK1 immune complex, and LURP1 promotes the formation of immune complexes and specific membrane localization of FLS2 and BAK1, enhances the activity of FLS2-BAK1, activates downstream molecules, and enhances the plant's immunity. Preferably, the upregulator of the light-controlled system includes: a reagent that increases LURP1 expression, a reagent that increases CRY1 expression, a reagent that promotes CRY1 activation of LURP1, a reagent that promotes N-terminal palmitoylation of LURP1, a reagent that promotes the localization of LURP1 to the cell membrane, or a reagent that promotes the interaction between LURP1 and FLS2. Preferably, the reagent for enhancing LURP1 expression includes: a recombinant LURP1 encoding gene or an expression construct containing the encoding gene; Preferably, the reagent that enhances CRY1 expression or promotes CRY1 activation of LURP1 includes: a recombinant CRY1 encoding gene or an expression construct containing the encoding gene.

6. Use of a blue light-based light control system or molecules therein in a plant as a molecular marker for identifying the plant's immune capacity; said light control system comprising CRY1 and its interacting protein LURP1; preferably, said light control system further comprising receptor-like kinase FLS2, forming a CRY1-LURP1-FLS2 pathway; more preferably, said light control system further comprising BAK1, wherein FLS2 and BAK1 form an FLS2-BAK1 immune complex.

7. A method for identifying plant immunity or for targeted screening of plants with high immunity, comprising: The operation of a blue light-based light control system in plants was analyzed; the light control system includes CRY1 and its interacting protein LURP1; preferably, the light control system also includes receptor-like kinase FLS2, forming a CRY1-LURP1-FLS2 pathway; more preferably, the light control system also includes BAK1, and FLS2 and BAK1 form an FLS2-BAK1 immune complex. If the plant exhibits high levels of CRY1 expression or activity, high levels of LURP1 expression or activity, high degree of N-terminal palmitoylation of LURP1, high amount of LURP1 located on the cell membrane, and strong interaction between LURP1 and FLS2, then the plant is a plant with high immune capacity.

8. A method for screening substances that enhance plant immunity, comprising: (1) Under blue light, the candidate substance is added to a system with a blue light-based light control system; the light control system includes CRY1 and its interacting protein LURP1; preferably, the light control system also includes receptor kinase FLS2 to form the CRY1-LURP1-FLS2 pathway; more preferably, the light control system also includes BAK1, and FLS2 and BAK1 form an FLS2-BAK1 immune complex; (2) Test the system and observe the operation of the light control system. If the expression or activity of CRY1 is increased, the expression or activity of LURP1 is increased, the degree of N-terminal palmitoylation of LURP1 is increased, the amount of LURP1 located in the cell membrane is increased, and the interaction between LURP1 and FLS2 is enhanced, it indicates that the candidate substance is a substance that can be used to enhance the plant's immune ability.

9. An isolated plant cell, tissue, or organ that recombinantly expresses a blue light-controlled system or an upregulator of said light-controlled system; said light-controlled system comprising: CRY1 and its interacting protein LURP1; preferably, the light-controlled system further includes receptor-like kinase FLS2 to form the CRY1-LURP1-FLS2 pathway; more preferably, the light-controlled system further includes BAK1, and the FLS2 and BAK1 form the FLS2-BAK1 immune complex; in the light-controlled system, LURP1 is recombinantly expressed, or CRY11 is recombinantly expressed, or FLS2 is recombinantly expressed.

10. The method or use as described in any one of claims 1-9, characterized in that, The plants mentioned include those selected from the group consisting of plants containing a blue light-based light control system; the light control system includes CRY1 and its interacting protein LURP1; preferably, the light control system further includes a receptor-like kinase FLS2, forming a CRY1-LURP1-FLS2 pathway.

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