Use of stir proteins to improve plant pattern-triggered immunity function and methods

Abstract

The present application provides the application and method of STIR protein in improving the function of plant pattern-triggered immunity. The STIR protein provided by the present application can effectively enhance the broad-spectrum resistance of plants to pathogens after being applied in plants to improve the expression level of STIR protein or enhance the activity of STIR protein, and has a good application prospect in crop disease-resistant breeding.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and more specifically, to the application and methods of STIR proteins in enhancing plant model-triggered immune function. Background Technology

[0002] Pattern-triggered immunity (PTI) is a core component of the plant immune system. It is triggered by pathogen-associated molecular patterns (PAMPs) such as the bacterial flagella-derived peptide flg22, the fungal cell wall component chitin, and the necrosis-inducing peptide nlp20. Once triggered, it elicits a series of immune responses and upregulates the expression of defense genes to limit pathogen infection and spread. Currently, most PTI enhancement strategies, such as overexpression of the flg22 receptor complex FLS2 or BAK1, while improving plant resistance to pathogens, often lead to inhibited plant growth, limiting their practical application.

[0003] Therefore, further research is needed to develop methods that can effectively improve plant pathogen resistance. Summary of the Invention

[0004] This application aims to address, to some extent, the technical problems existing in the prior art. To this end, this application proposes the application and method of STIR protein in enhancing plant model-triggered immune function. The STIR protein proposed in this application, when applied to plants to increase STIR protein expression levels or enhance STIR protein activity, can effectively enhance the broad-spectrum resistance of plants to pathogens. This provides theoretical support for research on the biological mechanisms of enhancing plant pathogen resistance and has promising application prospects in crop disease resistance breeding.

[0005] This application was made by the inventor based on the following discoveries: Plant-triggered immunity (PTI) activation triggers the production of calcium ions, including calcium ions (Ca). 2+ The process involves a series of early events, including influx of reactive oxygen species (ROS), a burst of reactive oxygen species (ROS), and phosphorylation of mitogen-activated protein kinases (MAPKs), as well as the upregulation of defense gene expression. However, the molecular mechanisms of PTIs are not fully elucidated, particularly regarding the functions and regulatory networks of downstream signaling components. TIR-only proteins, a conserved family of proteins in plants, lack the typical NB-ARC and LRR domains but possess a TIR (Toll / Interleukin-1 Receptor) domain. The NADase activity of the TIR domain has been shown to hydrolyze NAD. +It generates small molecule signals, but it is unclear whether TIR-only proteins regulate PTI through a similar mechanism.

[0006] Through experimental research, the inventors discovered that STIR1 and STIR2, as TIR-only proteins, undergo polymerization induced by flg22 signaling, promoting their NADase activity and efficiently hydrolyzing NAD. + The STIR1 and STIR2 proteins generate specific signaling molecules and activate the EDS1-PAD4-ADR1 signaling pathway, promoting downstream immune responses of PTI and enabling plants to acquire broad-spectrum resistance to pathogens. The elucidation of the mechanism of STIR1 and STIR2 proteins in pattern-triggered immunity, which differs from known TNL or CNL models, fills the functional gap of TIR-only proteins in PTI. Furthermore, it reveals the conservation of the immune function of STIR homologous proteins in various plants such as rice, wheat, soybean, tomato, and Arabidopsis thaliana. This provides a solid theoretical foundation for developing small molecule agonists or expression vectors targeting the STIR1 / 2 pathway, or for knocking out negative regulators of STIR1 / 2 or enhancing their endogenous expression through gene editing technology to achieve spatiotemporally controllable immune activation in transgenic crops.

[0007] Based on this, in a first aspect of this application, the application of STIR proteins in enhancing plant pattern-triggered immune function is proposed. According to embodiments of this application, the STIR proteins include at least one of the following: STIR1 protein or a functional analogue thereof, STIR2 protein or a functional analogue thereof. The inventors have discovered that activation of STIR1 and STIR2, or analogues of both, can effectively enhance plant pattern-triggered immune function, enabling plants to acquire broad-spectrum resistance to a variety of pathogens.

[0008] According to embodiments of this application, the plant includes at least one of Arabidopsis thaliana, tomato, soybean, rice, and Nicotiana benthamiana.

[0009] According to embodiments of this application, the STIR protein is derived from *Nicotiana benthamiana*.

[0010] According to embodiments of this application, the STIR1 protein has an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 75% identity with it. In some embodiments, the amino acid sequence used has more than 99%, more than 95%, more than 90%, more than 85%, more than 80%, or more than 75% identity with the STIR1 protein. The STIR1 protein that satisfies the above amino acid sequence can effectively improve the pattern-triggered immune function of plants, enabling plants to acquire broad-spectrum resistance to a variety of pathogens.

[0011] MAEAKEENREKNEEEESVKLFVGQIPKHMSESQLLTLFQEFAVVDEVNIIKDKITRASRGCCFLLCPSREEADKLVNACHNKKTLPGANSLLQVKYADGELERLEHKLFVG MLPKNVSEAEVQSLFSKYGTIKDLQILRGAQQTSKGCAFLKYETKEQAVSAMESINGKHKMEGSTVPLVVKWADTERERHTRRLQKAQSHIARLGNGDPTNPSLFGALPMG YVPPYNGYGYHQPPGTYGYMLPPIQNQAAFSNMIAQPNQGNNNALQGTSPDSVPPRLARRNFPMPPGNYMGSGYPAMRGHPFPFAYPRGIVSPRPLSSSPGSISPGMSTPL GIGLSSVVQTEGPEGANLFIYNIPREFGDQELAAAFQSFGIVLSAKVFVDKATGVSKCFGFVSYDSQAAAQNAIDMMNGRHLGGKKLKVQLKRDSNNGQPSSNPSLIS (SEQ IDNO: 1).

[0012] According to embodiments of this application, the STIR2 protein has an amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence having at least 75% identity with it. In some embodiments, the amino acid sequence used has more than 99%, more than 95%, more than 90%, more than 85%, more than 80%, or more than 75% identity with the STIR1 protein. The STIR2 protein that satisfies the above amino acid sequence can effectively improve the pattern-triggered immune function of plants, enabling plants to acquire broad-spectrum resistance to a variety of pathogens.

[0013] MAELQREEERQEEEQQSEESVKLFVGQVPKHMTESQLVEMFQEFAIVDEVNIIKDKTTRASRGCCFVICPSREEADKAVNACHNKKTLSGASSPLQVKYADGELERLEHK LFVGMLPKNVSDPEVSALFSQYGVIKDLQILRGSQQTSKGCAFLKYEKKEQAVAAIDALHGKHKMEGATVPLVVKWADTEKERQARRAQKSLSHASDSRQHPSLFGALPM GYMPPYNGYGYQTPGAYGLMQYRLPSMQNQSAFQNIVPPINQASALRGGAPDLSPGISPRNYAMSPGSYGSAYPAVPGIQYSMPYPGGVMNTRPPSGSPGSIPPSTTNSH SAASSSVSSSTGGQVEGPPGANLFIYHIPQEFGDQELANAFQPFGRVLSAKVFVDKATGVSKCFGFVSYDSTAAAQTAISMMNGCQLGSKKLKVQLKRDNKQNKHY (SEQ ID NO: 2).

[0014] According to embodiments of this application, the gene encoding the STIR1 protein has a nucleotide sequence as shown in SEQ ID NO: 3 or a nucleotide sequence having at least 80% identity with it. In some embodiments, the nucleotide sequence used has more than 99%, more than 95%, more than 90%, more than 85%, or more than 80% identity with the gene encoding the STIR1 protein. The gene encoding the above-mentioned nucleotide sequence can effectively express the STIR1 protein, improve the plant's pattern-triggered immune function, and enable the plant to acquire broad-spectrum resistance to a variety of pathogens.

[0015]

[0016] According to embodiments of this application, the gene encoding the STIR2 protein has a nucleotide sequence as shown in SEQ ID NO: 4 or a nucleotide sequence having at least 80% identity with it. In some embodiments, the nucleotide sequence used has more than 99%, more than 95%, more than 90%, more than 85%, or more than 80% identity with the gene encoding the STIR2 protein. The gene encoding the above-mentioned nucleotide sequence can effectively express the STIR2 protein, improve the pattern-triggered immune function of plants, and enable plants to acquire broad-spectrum resistance to a variety of pathogens.

[0017]

[0018] In a second aspect of this application, a plant-based model-triggered immune function activator is proposed. According to embodiments of this application, the activator includes at least one of the STIR1 protein or a functional analog thereof, the STIR2 protein or a functional analog thereof, the gene encoding the STIR1 protein, and the gene encoding the STIR2 protein.

[0019] According to embodiments of this application, the activator further comprises an agriculturally acceptable carrier.

[0020] In a third aspect of this application, the activator described in the second aspect embodiment is used in the preparation of a plant immune enhancer, which, according to the embodiments of this application, is used to enhance plant model-triggered immune function to prevent and control plant diseases.

[0021] In a fourth aspect of this application, a method for enhancing plant-based immune function is proposed. According to an embodiment of this application, the method includes: upregulating the expression of STIR proteins in plants or increasing the activity of STIR proteins in plants; the STIR proteins include at least one of the following: STIR1 protein or a functional analog thereof, STIR2 protein or a functional analog thereof.

[0022] According to embodiments of this application, the STIR1 protein in the method has an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 75% identity with it; the STIR2 protein in the method has an amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence having at least 75% identity with it.

[0023] According to embodiments of this application, the plant in the method includes at least one of Arabidopsis thaliana, tomato, soybean, rice, and Nicotiana benthamiana.

[0024] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0025] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 The materials used in constructing the mutant material in Example 1 of this application pHEE4011E-NbSTIR1 / 2 Atlas of gene editing vectors; Figure 2 Two independent structures constructed for Embodiment 1 of this application stir1 stir2 Mutation patterns in double mutant plant lines; Figure 3 Sequence alignment diagram of STIR1 / 2 protein with other related TIR-only proteins; Figure 4 Phylogenetic tree of STIR1 / 2 proteins and other related TIR-only proteins; Figure 5 Wild-type and wild-type plants were cultured in a plant culture room for 4 weeks. stir1 stir2 Phenotypic diagram of plant growth and development of double mutants; Figure 6 Wild type and stir1 stir2 Statistical chart of pathogen quantity after inoculation of double mutant plants with pathogen and phenotypic chart of plant disease symptoms; Figure 7 Wild type and stir1 stir2 Statistical chart of pathogen counts after double mutant plants were inoculated with different pathogens; Figure 8 A statistical chart showing the number of pathogens after instantaneous transformation and re-inoculation with different pathogens. Detailed Implementation

[0026] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0027] It should be noted that in the embodiments of this application, the terms "comprising" or "including" are open-ended expressions, that is, they include the content specified in this application, but do not exclude other aspects.

[0028] In the embodiments of this application, the terms "optionally," "optionally," or "optionally" generally refer to events or conditions described subsequently that may but may not occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0029] In this application embodiment, the term "pattern-triggered immunity" refers to a series of innate immune defense responses triggered by plants through pattern recognition receptors (PRRs) on their cell membranes, which specifically recognize pathogen-associated molecular patterns (PAMPs) or microbial-associated molecular patterns (MAMPs) of pathogenic microorganisms.

[0030] In the embodiments of this application, the term "functional analogue" refers to a substance that may have a different chemical structure but can perform the same or similar biological / physicochemical functions as STIR1 or TIR2 proteins in plants.

[0031] In the embodiments of this application, the term "identity" refers to the proportion / number of identical nucleotide / amino acid residues appearing at the same position in two or more homologous nucleic acid / amino acid sequences. It is an important indicator for quantifying the similarity between sequences, taking into account the complete matching of residues, and does not include conserved substitutions.

[0032] In this application embodiment, the term "agriculturally acceptable carrier" refers to a carrier material / formulation system that is suitable for agricultural production scenarios, can carry and deliver active ingredients (pesticides, fertilizers, microbial agents, biostimulants, genes or proteins, etc.), and complies with agricultural safety standards and has no adverse effects on the target crop / environment.

[0033] In the embodiments of this application, the term "NB-ARC" refers to the core functional domain present in NLR-type immune receptors. It is the core module for NLR receptors to achieve nucleotide binding, conformational switching and immune signal activation. It is widely present in the innate immune receptors of plants and animals and plays an important regulatory role in plant disease resistance immunity (ETI).

[0034] In the embodiments of this application, the term "LRR" is an abbreviation for Leucine-Rich Repeat, which is a type of tandem repeat domain composed of alternating conserved motifs rich in leucine and variable sequences. It is widely found in various proteins of animals, plants, and microorganisms, and is a core functional domain in plant NLR disease resistance receptors and pattern recognition receptors (PRRs).

[0035] In this embodiment, the term "TNL" is an abbreviation for TIR-NB-ARC-LRR, which is one of the classic subtypes of plant NLR (nucleotide-binding domain and leucine-rich repeat sequence) disease-resistant immune receptors. It is composed of a TIR domain, an NB-ARC domain, and an LRR domain connected in series from the N-terminus to the C-terminus. It is the core intracellular receptor for effector-triggered immunity (ETI) mediated by dicotyledonous plants. By specifically recognizing pathogen effectors and initiating downstream immune signaling pathways, it achieves precise defense against pathogens.

[0036] In this embodiment, the term "CNL" is an abbreviation for CC-NB-ARC-LRR, which is one of the core subtypes of plant NLR (nucleotide-binding domain and leucine-rich repeat sequence) disease-resistant immune receptors. It is one of the two major types of plant NLR receptors, along with TNL. It is composed of a CC (coil-and-coil) domain, an NB-ARC domain, and an LRR domain connected in series from the N-terminus to the C-terminus. It is an intracellular disease-resistant receptor widely distributed in dicotyledonous and monocotyledonous plants, mediating effector-triggered immunity (ETI). By specifically recognizing pathogen effectors, it initiates downstream immune signals to achieve defense against various pathogens such as bacteria, fungi, and viruses. It is also a key NLR type in monocotyledonous plants that replaces TNL in exercising disease resistance.

[0037] In the embodiments of this application, the terms "STIR1 / 2" and "STIR1 and STIR2" refer to the proteins or encoding genes of STIR1 and STIR2 that have the same or substantially the same meaning.

[0038] In the embodiments of this application, the term "NbSTIR1 / 2" refers to the proteins or encoding genes of STIR1 and STIR2 in Nicotiana benthamiana.

[0039] In the embodiments of this application, the term "competent state" refers to the physiological state in which microbial cells (bacteria, fungi, etc.) are able to actively take up exogenous nucleic acids (plasmids, DNA fragments) from the surrounding environment and achieve their stable existence / expression within the cell. It is a special physiological phenotype of microorganisms and a core prerequisite for achieving exogenous gene transformation in genetic engineering.

[0040] In the embodiments of this application, the term "potency" is an important quantitative indicator that characterizes the bioactivity intensity of a bioactive substance and reflects the effective activity level of the bioactive substance.

[0041] In the embodiments of this application, the term "knockout mutation" refers to a type of gene mutation in which the coding / functional region of a specific gene in the genome is deleted, inserted, or replaced by a base through artificial directed manipulation, resulting in the complete termination of transcription and translation of the gene or the loss of all biological functions of the expression product, ultimately achieving complete inactivation of the target gene.

[0042] In this application embodiment, the term "sgRNA" is an abbreviation for Single Guide RNA, which is a core component of the CRISPR / Cas9 gene editing system. It is an artificially designed fusion single-stranded RNA molecule, which is formed by the fusion of crRNA (CRISPR RNA) that targets the target gene and tracrRNA (trans-activating crRNA) that binds to the Cas9 nuclease through base complementarity or artificial linkage. Its core function is to accurately target and recognize the target DNA sequence in the genome and guide the Cas9 nuclease to that site for double-strand cutting.

[0043] In this embodiment, the term "leaf disc method" refers to a classic and simple Agrobacterium-mediated transformation method in plant genetic transformation. By cutting plant leaves into small discs (leaf discs), the exogenous target gene is introduced into the leaf disc cells using the infection characteristics of Agrobacterium. Then, through plant tissue culture, the leaf discs undergo dedifferentiation and redifferentiation, ultimately obtaining complete transgenic plants. This method is suitable for the genetic transformation of dicotyledonous plants and is one of the most commonly used techniques in crop genetic engineering and disease resistance breeding.

[0044] In this application embodiment, the term " stir1 stir2A "double mutant" refers to a mutant in which the coding gene loci of STIR1 and the coding gene loci of STIR2 in the same plant individual / cell undergo stable mutations (knockout, point mutation, deletion, etc.) through artificial directed manipulation, and both mutant phenotypes can be stably inherited and expressed.

[0045] In this application embodiment, the term "frameshift" refers to a type of gene mutation in which the insertion or deletion of bases in the coding region of a gene (the number of inserted / deleted bases is not an integer multiple of 3) causes an irreversible shift in the reading frame of all codons after the mutation site, ultimately resulting in a complete change in the amino acid sequence of the translated protein and the loss of its biological function.

[0046] In the embodiments of this application, the term "overexpression mutation" refers to a gene modification type in which the transcription / translation level of a target gene in the genome of an organism is significantly higher than that of the wild type through artificially directed manipulation.

[0047] In this application, the term "point mutation" refers to a precise mutation type in protein function and gene editing research, which involves the targeted substitution of 1-2 bases in a single codon in a gene coding region or the site-specific deletion / insertion of a single residue, resulting in the substitution of a specific amino acid residue in the translated protein polypeptide chain. This causes the protein encoded by the gene to lose its function without changing the sequence of subsequent residues. It is an important technical means to analyze the function of specific residues in a protein and to target and modify the structure and activity of a protein.

[0048] In this application embodiment, the term "ETI" is an abbreviation for Effector-Triggered Immunity, which is a defense strategy in the plant's innate immune system following PTI. Plants use intracellular NLR-type immune receptors (TNL / CNL) to specifically recognize effectors (Avr proteins) secreted by pathogens, thereby initiating a high-intensity, specific immune response. Ultimately, through hypersensitivity response (HR) and systemically acquired resistance (SAR), plants achieve precise defense against pathogens, which is an important immune mechanism for plants to resist pathogen infection.

[0049] In the embodiments of this application, the term "linearized vector" refers to the process and product of cutting a circular plasmid vector with a restriction endonuclease to transform it from a closed circular shape into a linear double-stranded DNA molecule, so as to facilitate the ligation of exogenous fragments or improve the integration efficiency of host cells.

[0050] The technical solutions in the embodiments of this application will be described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0051] Example 1: Construction of STIR1 / 2 mutant material 1. Experimental reagents and materials Wild-type Nicotiana benthamiana plants were cultured for 4 weeks after sowing for later use; pHEE4011E vector (containing Cas9 expression cassette, hygromycin resistance selection marker, and Arabidopsis U6-26 promoter) and pE1776 vector (containing octopine synthase promoter for transient expression in tobacco); restriction endonuclease BsaⅠ-HF (20 U / μL, purchased from NEB) and T4 DNA ligase (400 U / μL, purchased from Thermo Fisher); Agrobacterium GV3101 strain (competent cells, titer ≥1×10⁻⁶). 8 CFU / μL); specific primers suitable for different experimental purposes (all at a concentration of 10 μmol / L, synthesized by Sangon Biotech Co., Ltd.); hygromycin (purity ≥98%, prepared as a 50 mg / mL stock solution, filtered, sterilized, and then aliquoted for storage); DNA extraction kit (commercially available conventional plant genomic DNA extraction kit), PCR amplification kit (2×Taq Master Mix), etc.

[0052] 2. Construction of mutant materials 2.1 Construction of STIR1 / 2 knockout mutant materials (1) sgRNA design and vector construction: Based on the conserved TIR domain sequences of the coding regions of NbSTIR1 (SEQ ID NO: 3) and NbSTIR2 (SEQ ID NO: 4), specific sgRNAs were designed, and BsaI restriction sites were introduced at both ends of the sgRNAs. 1 μg of pHEE4011E vector was taken, 1 μL of BsaI-HF enzyme and 2 μL of 10×CutSmart Buffer were added, and sterile double-distilled water was added to a final volume of 20 μL. The mixture was digested at 37 ℃ for 1 h, and the linearized vector fragment (approximately 13 kb) was recovered by agarose gel electrophoresis. 5 μL of each pair of sgRNA primers (the upstream and downstream primers for NbSTIR1 / 2 related sgRNAs are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively) were added, 2 μL of 10×PCR Buffer was added, and the mixture was denatured at 95 ℃ for 5 min and then allowed to cool naturally to room temperature to form double-stranded sgRNA (ds-sgRNA). 50 ng of linearized vector and 100 ng of ds-sgRNA were added, along with 1 μL of T4 DNA ligase and 2 μL of 10×T4 Ligase Buffer. Sterile water was added to a final volume of 20 μL, and the ligation reaction was carried out overnight at 16 °C to construct the pHEE4011E-NbSTIR1 / 2 dual-target gene editing vector. 5 μL of the ligation product was transformed into *E. coli* DH5α competent cells. Single colonies were picked, cultured, and the plasmid was extracted. BsaI restriction enzyme digestion and Sanger sequencing confirmed that the vector's sgRNA sequence was free of mutations and that the insertion direction was correct. (Reference) Figure 1 As shown, the Cas9 expression cascade, U6 promoter, sgRNA insertion site, and the location of the hygromycin resistance gene Hyg are marked in the constructed pHEE4011E-NbSTIR1-NbSTIR2 vector map.

[0053] ATATATGGTCTCGATTGACACAAAAAGAAATGTAGCGTTTTAGAGCTAGAAATAGC (SEQ ID NO: 5); ATTATTGGTCTCGAAACAATTTTACAATTGCGAATCCAATCTCTTAGTCGACTCTAC (SEQ ID NO: 6).

[0054] (2) Agrobacterium-mediated transformation of Nicotiana benthamiana: 2 μg of the constructed recombinant vector plasmid was added to 100 μL of Agrobacterium GV3101 competent cells. After incubating on ice for 30 min, the cells were rapidly frozen in liquid nitrogen for 5 min, heat-shocked in a water bath at 37 ℃ for 5 min, and then incubated on ice for 2 min. 500 μL of antibiotic-free LB medium was added, and the cells were cultured at 28 ℃ and 200 rpm for 2 h with shaking. The culture was then spread on LB plates containing 50 mg / L rifampicin and 50 mg / L gentamicin, and cultured at 28 ℃ for 48 h. Positive single colonies were picked and expanded. Four-week-old wild-type Nicotiana benthamiana plants were transformed using the leaf disc method. Functional leaves free from diseases and pests were selected, and leaf discs were prepared using a sterile punch (0.8 cm in diameter). The leaf discs were then immersed in Agrobacterium bacterial solution (OD200). 600 Infect the leaf discs with 0.6 mg / L 6-BA for 10 min, gently shaking them during this time. Remove the leaf discs and blot the surface bacterial solution with sterile filter paper. Inoculate them onto MS medium (containing 0.1 mg / L 6-BA and 0.05 mg / L NAA) and incubate in the dark at 25 °C for 2 days. Then transfer the leaf discs to selection medium (MS medium + 0.1 mg / L 6-BA + 0.05 mg / L NAA + 25 mg / L hygromycin + 250 mg / L cephalosporin). Change the medium every 2 weeks to obtain T0 generation resistant regenerated plants. Transplant the plants into nutrient soil when they have grown 4-5 true leaves.

[0055] (3) stir1 stir2Double mutant identification: DNA was extracted from young leaves of T0 generation positive plants using a plant genomic DNA extraction kit (0.1 g of leaves per sample, ground and processed according to the kit instructions, with a final elution volume of 50 μL). Specific identification primers covering the sgRNA target region were designed (the upstream and downstream identification primers for NbSTIR1 are shown in SEQ ID NO: 7 and SEQ ID NO: 8, and the upstream and downstream identification primers for NbSTIR2 are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively). The amplified fragment length was approximately 500 bp. The PCR amplification system was 20 μL: 10 μL of 2×Taq Master Mix, 0.8 μL of each primer, 2 μL of DNA template, and 6.4 μL of sterile water. The amplification program was: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ final extension for 5 min. After PCR products were detected by 1.5% agarose gel electrophoresis, the target fragment was recovered and ligated into the pMD19-T vector (TA cloning vector, purchased from Takara). This vector was then transformed into *E. coli* DH5α, and three single colonies were selected for sequencing. Plants exhibiting simultaneous NbSTIR1 and NbSTIR2 gene editing were screened, and T1 generation seeds were harvested through self-pollination. After sowing, homozygous lines were selected using 25 mg / L hygromycin, ultimately yielding two independent double mutant lines. stir1-1 stir2-1 , stir1-2 stir2-2 ). refer to Figure 2 The sequencing results shown indicate that strain 1 ( stir1-1 stir2-1) The NbSTIR1 gene has an 8 bp deletion (located at amino acids 120-122 of the TIR domain), and the NbSTIR2 gene has a 5 bp deletion; line 2 ( stir1-2 stir2-2 A 1 bp insertion occurred in the NbSTIR1 gene and a 1 bp insertion occurred in the NbSTIR2 gene, both resulting in frameshifts and preventing the synthesis of functional STIR proteins.

[0056] CAAGAGCGGGCAAGGCATT (SEQ ID NO: 7); CTGCATTATTGCATCCGAAGCTTT (SEQ ID NO: 8); GGCTGCTCATATATTGAATAGTCATATTCGACTT (SEQ ID NO: 9); GCTTGTTGTAAGGCTCCGGAT (SEQ ID NO: 10).

[0057] 2.2 Construction of STIR1 overexpression and point mutation vectors (1) Design and construction of STIR1 overexpression vector: Homologous recombination primers were designed based on the TIR domain sequence of the coding region of NbSTIR1 (SEQ ID NO: 3) gene (the upstream and downstream primers for recombination are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively). Tobacco cDNA was used as a template for PCR amplification. The fragment was excised and recovered. 1 μg of pE1776 vector linearized by double digestion with KpnI and XbaI was added, along with 1 μL of homologous recombinase Exnase II, 2 μL of 5×CE II Buffer, 1 μg of PCR fragment, and sterile double-distilled water to 10 μL. After incubation at 37 ℃ for 0.5 h, the vector was transformed into Escherichia coli DH5α competent cells. Single colonies were picked, cultured, and plasmids were extracted. The correct vector construction was confirmed by Sanger sequencing.

[0058] caaatcgactctaggggtaccATGCAACGTTCAGCAATATCTTCC (SEQ ID NO: 11); gtatgggtaagcagctctagaATAGTTGTGTAAAAGTTGTTTCGACG (SEQ ID NO: 12).

[0059] (2) Design and construction of STIR1 enzyme activity point mutation vector: Based on the sequence obtained from the above amplification as a template, mutation primers were designed at the mutation site (the upstream and downstream primers for the point mutation are shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively). The mutated STIR1 gene fragment was obtained by bridging PCR. The construction and verification steps were similar to those described above. The obtained point mutation material is referred to as STIR1 in the following description and in the images. E109A express.

[0060] CTTGCATgcgTTGTCTCTGATGATGGAATTAAAGAAG (SEQ ID NO: 13); GAGACAAcgcATGCAAGCAAAAATAAGAATCACAA (SEQ ID NO: 14).

[0061] Example 2: Application potential of STIR1 / 2 in various plants Phylogenetic tree construction and sequence alignment of STIR1 / 2-related TIR-only proteins.

[0062] 1. Sequence collection and alignment Amino acid sequences of proteins to be compared were collected. All sequences were obtained from the esembl plant database. The sequence selection criteria included: sequence integrity ≥95%, containing only a single TIR domain, and having no other functional domains. The amino acid sequences to be compared included, but were not limited to: Arabidopsis thaliana AtTX3, AtTX9, AtRBA1, rice OsTIR, brachypodium bdTIR, barley HyTIR, and STIR1 (SEQ ID NO: 1, referred to as NbSTIR1 in the following description and in the images) and STIR2 (SEQ ID NO: 2, referred to as NbSTIR2 in the following description and in the images) derived from tobacco in this application.

[0063] The collected amino acid sequences to be aligned were processed using MEGA11 software (version 11.0.13). Multiple sequence alignment was performed using ClustalX2 software (version 2.1) with the following parameters: Gap Opening Penalty of 10.0, Gap Extension Penalty of 0.2, amino acid substitution matrix of BLOSUM62, and percentage of delayed divergent sequences of 30%. Iterative alignments were performed until the results stabilized. After alignment, the results were manually corrected using MEGA11 software to remove inconsistent gap regions, ultimately obtaining consistent aligned sequences to ensure the accuracy and reliability of the sequence alignment. Some sequence alignment results are referenced below. Figure 3 As shown.

[0064] 2. Phylogenetic tree construction Based on the corrected multiple sequence alignment results, a phylogenetic tree was constructed using the Neighbor-Joining (NJ) method with the following parameter settings: the substitution model was Poisson Correction to account for the unevenness of amino acid substitutions; the bootstrap value was set to 1000 repetitions, and the branch reliability of the tree was evaluated by random resampling; pairwise deletion was used to handle missing data to avoid data bias; other parameters remained at the software default values. Simultaneously, a Maximum Likelihood (ML) tree was constructed as a control to verify the reliability of the NJ tree. The ML tree parameters were set as follows: the substitution model was JTT+G+I, and the bootstrap value was 1000 repetitions.

[0065] The developmental tree structure was optimized. FigTree software was used to enhance the constructed NJ tree, adjusting branch line widths and labeling the species origin, GenBank accession number, and TIR domain characteristic sites for each protein. Tree structure analysis clarified the evolutionary relationship and taxonomic position of NbSTIR1 / 2 with other TIR-only proteins.

[0066] refer to Figure 4As shown, NbSTIR1 and NbSTIR2 cluster closely together, belonging to a TIR-only protein subfamily different from the Arabidopsis immune protein AtRBA1. Multiple sequence alignment results show that the core active site (conserved amino acid residue Glu) of the TIR domain in NbSTIR1 and NbSTIR2 is completely identical to other TIR-only proteins. This branch is adjacent to the branches containing Arabidopsis AtTX3 and AtTX9, as well as TIR-only proteins in monocotyledons (rice, Brachypodium clavatum, and barley), indicating a close evolutionary distance. This suggests that STIR1 / 2 exhibits high evolutionary conservation in both monocotyledons and dicotyledons, providing evolutionary evidence for the functional conservation of STIR1 / 2 in other plants. It also lays the foundation for elucidating the evolutionary patterns and functional differentiation of TIR-only proteins and demonstrates the potential application of STIR1 / 2 in various plants.

[0067] Example 3: The regulatory effect of STIR1 / 2 on plant model-triggered immune responses 1. Experimental materials 4-week-old Benedictine wild-type tobacco (WT) and stir1 stir2 Double mutant plants ( stir1-1 stir2-1 , stir1-2 stir2-2 Culture conditions: 16 h light / 8 h dark, temperature 25±1℃, humidity 70±5%, light intensity 200 μmol·m - ²·s - ¹; *Pseudomonas syringae* tomato pathogenic strain Pst DC3000 ΔHopQ1 strain (lacking the HopQ1 gene, unable to activate the ETI reaction in *Nicotiana benthamiana*); LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0, autoclaved at 121°C for 20 min); rifampin (purity ≥98%, prepared as a 50 mg / mL stock solution, filtered and sterilized, final concentration 50 mg / L); 10 mM MgCl2 solution (0.203 g of MgCl2·6H2O was dissolved in 100 mL of sterile water and autoclaved at 121°C for 20 min); sterile water; sterile perforator (0.8 cm in diameter, corresponding area 0.5 cm²). 2 Equipment includes: 1.5 mL centrifuge tubes, pipettes (range 10 μL-1000 μL), constant temperature shaking incubator, constant temperature incubator, ImageJ software (version 1.8.0), etc.

[0068] 2. Experimental Methods (1) Pathogen culture From -80℃ refrigerator Pst50 μL of the DC3000 ΔHopQ1 strain glycerol cryopreservation solution was inoculated into 5 mL of LB medium containing 50 mg / L rifampicin and cultured overnight (approximately 16 h) at 28 °C with shaking at 200 rpm until the bacterial culture reached OD500. 600 The value reached 1.0-1.2. Take 1 mL of overnight culture and add 10 mL of fresh LB medium containing rifampin, continue to culture with shaking until OD reaches 1.0-1.2. 600 =0.6, centrifuge at 8000 rpm for 5 min to collect bacterial cells, discard the supernatant, resuspend the bacterial cells in 10 mM MgCl2 solution, wash twice, and finally adjust the bacterial concentration to OD. 600 =1×10 5 (Verified by serial dilution plating, the colony count was 1×10⁻⁶) 5 (CFU / mL), keep on ice for later use to avoid cell inactivation.

[0069] (2) Injection and pathogen counting Select the 3rd-4th functional leaves (counting upwards from the base) of WT and double mutant plants, injecting 3 leaves per plant, with 3 replicate sites per leaf (site spacing ≥2 cm, avoiding veins). Use a needleless syringe injection method, injecting 50 μL of adjusted bacterial suspension at each site, ensuring complete penetration of the bacterial suspension into the leaf tissue without spillage. The WT inoculated group served as the control group, denoted as Nb. After inoculation, the plants were cultured under normal conditions. Five days later, leaf tissue (0.8 cm in diameter, 0.5 cm² in area) was collected from each inoculation site using a sterile punch. 2 Place the bacterial suspension in a 1.5 mL centrifuge tube containing 500 μL of 10 mM MgCl2 solution, add 3 sterile glass beads (3 mm in diameter), and grind on a tissue homogenizer for 30 s (30 Hz) to prepare a bacterial suspension. Then, perform a 10-fold serial dilution (10... 0 10¹, 10², and 10³ times dilutions were used. 100 μL of each dilution was spread onto LB agar plates containing 50 mg / L rifampicin. Three replicates were created for each dilution. After incubation at 28°C for 48 h, the number of colonies on the plates was counted (only plates with colony counts between 30 and 300 were counted). The number of pathogens per square centimeter of leaf tissue (CFU / cm²) was calculated. 2 The average and standard deviation of pathogen reproduction for each strain were calculated.

[0070] (3) Spray inoculation and phenotypic observation: The concentration (OD) was adjusted. 600 =1×10 5 )of PstDC3000 ΔHopQ1 bacterial suspension was added to a small sprayer and sprayed evenly onto the leaf surface of WT and double mutant plants, with a spray volume of 5 mL per plant, ensuring that both the upper and lower surfaces of the leaves were completely covered by the bacterial suspension without any obvious droplets. Immediately after inoculation, the plants were covered with transparent plastic wrap and placed in a culture room for 24 hours of humidified culture (maintaining humidity of 90%±5%, with other conditions unchanged). Then, the plastic wrap was removed, and the plants were transferred to normal culture conditions for another 4 days, for a total of 5 days after inoculation. The phenotypic characteristics of the plant leaves were photographed using a digital camera (focal length 50 mm, aperture f / 8, shutter speed 1 / 125 s). The 3rd-4th functional leaves of each plant were selected for photography, with a total of 10 leaves per plant system. The photos were opened with ImageJ software, the scale bar was set, and the lesion areas and the total leaf area were manually outlined. The proportion of lesion area to the total leaf area was calculated, and the average and standard deviation of lesion coverage rate for each line were statistically analyzed.

[0071] (3) Statistical analysis Data processing was performed using SPSS 26.0 software. Normality tests (Shapiro-Wilk test) and homogeneity of variance tests (Lvene test) were first conducted to confirm that the data conformed to a normal distribution and homogeneity of variance. Then, an independent samples t-test was used to compare the differences in pathogen proliferation and lesion area between the WT and double mutant lines. A significance level of α = 0.05 was set, with P < 0.05 considered significant. Data results are expressed as mean ± standard deviation. Significant differences were marked with an asterisk in the figures (* indicates P < 0.05, ** indicates P < 0.01). Three biological replicates were set up, each containing 10 plants, to ensure the reliability and reproducibility of the experimental results.

[0072] 3. Experimental Results refer to Figure 5 As shown, under the same normal culture conditions, 4-week-old... stir1 stir2 The double mutants (two lines) showed no significant differences from the WT plants in terms of plant type, leaf morphology, leaf color, and number of branches. Both exhibited robust plant type, bright green leaves, and well-developed leaves, without any abnormal phenotypes such as yellowing, dwarfing, or deformity.

[0073] refer to Figure 6 As shown in Figure A, the pathogen count results indicate that... stir1 stir2 In the leaves of the two mutant lines Pst The colony counts of DC3000 ΔHopQ1 were significantly higher than those of WT plants, with a highly statistically significant difference (P<0.01). Furthermore, there was no significant difference between the two mutant lines, indicating that the mutant phenotype was stable. (Reference) Figure 6 As shown in Figure B, the disease phenotype indicates that the leaf lesion area of ​​the double mutant plants is significantly larger than that of the WT. stir1 stir2The double mutant strains showed more severe symptoms when infected by the pathogen, with obvious yellowing and necrotic areas on the leaves, while the WT leaves only showed a few small lesions.

[0074] The above results indicate that after the STIR1 / 2 gene is deleted, the plant's response to... Pst The basal immune defense capacity of DC3000 ΔHopQ1 was significantly reduced, and pathogens were more likely to multiply and spread in the leaves, confirming that STIR1 / 2, as a positive regulator, participates in the regulation of the plant's basal immune response.

[0075] Example 4: The regulatory effect of STIR1 / 2 on plant immune sensitization function 1. Experimental materials 4-week-old Benzodiazepines WT, stir1 stir2 Double mutant (plant culture conditions same as in Example 3); flg22 peptide (22 amino acid fragments at the N-terminus of bacterial flagellin, sequence QRLSTGSRINSAKDDAAGLQIA (SEQ ID NO: 15), purity ≥95%, purchased from Jier Biochemical Co., Ltd.); Pst DC3000 ΔHopQ1 and Xe 85-10 ΔXopQ strain (lacking the XopQ gene, unable to activate the ETI reaction in Tobacco Benzovia); 10 mM MgCl2 solution; sterile syringe (1 mL, needleless), sterile centrifuge tubes, pipettes, constant temperature shaking incubator, constant temperature incubator, SPSS 26.0 software, etc.; sterile water (as a mock control treatment solution).

[0076] 2. Experimental Methods 2.1 Regulatory effect on PTI immune sensitization function of stable transgenic plants (1) flg22 preprocessing Dissolve flg22 peptide in sterile water to prepare a 1 μM flg22 working solution (accurately weigh 0.022 mg flg22 peptide, add 10 mL sterile water, dissolve thoroughly, aliquot, and store at 4℃ for later use; shelf life 24 h). Select the 3rd-4th functional leaves of WT and double mutant plants. On each leaf, set 2 treatment sites (flg22 pretreatment sites) and 2 control sites (mock sites, with sterile water as the control mock), with a spacing of ≥2 cm between sites, avoiding leaf veins. Using a 1 mL sterile syringe without a needle, inject 50 μL of 1 μM flg22 solution into each flg22 pretreatment site and 50 μL of sterile water into each mock control site, ensuring complete penetration of the treatment solution into the leaf tissue. After pretreatment, place the plants in their original culture conditions for routine culture, avoiding leaf damage during this period to ensure stable pretreatment effects.

[0077] (2) Inoculation with pathogens One day after flg22 pretreatment, 50 μL of the solution was injected into the flg22 pretreatment site and the mock control site on the same leaf. Pst DC3000 ΔHopQ1 bacterial solution or Xe 85-10 ΔXopQ bacterial suspension (concentration OD) 600 =1×10 5 The preparation method was the same as in Example 3), and the inoculation operation was consistent with Example 3, ensuring that the inoculation amount and depth at each site were consistent. After inoculation, the plants were placed under the original culture conditions for routine culture, and the environment was kept stable to avoid temperature and humidity fluctuations affecting pathogen reproduction. Three biological replicates were set up, each containing 10 plants, to ensure the reliability of the experimental data.

[0078] (3) Pathogen count and statistics Five days after inoculation, leaf tissue (0.8 cm in diameter, 0.5 cm² in area) was collected from each treatment site and control site. 2 The bacterial suspension was prepared by grinding, serially diluted 10-fold, spread on plates, and after incubation, the number of pathogens per square centimeter of leaf tissue (CFU / cm²) was counted. 2 The inhibition rate of flg22 pretreatment on pathogen reproduction was calculated, where the inhibition rate = (number of pathogens in the mock control group - number of pathogens in the flg22 pretreatment group) / number of pathogens in the mock control group × 100%. Statistical analysis was performed using SPSS 26.0 software. Normality and homogeneity of variance tests were performed first, followed by two-way ANOVA to compare the differences between WT and double mutants, and between flg22 pretreatment and mock controls. P < 0.05 was considered statistically significant. Data are expressed as mean ± standard deviation.

[0079] 3. Experimental Results refer to Figure 7 As shown in Figure A, which depicts the vaccination process. Pst Pathogen statistics after DC3000 ΔHopQ1, Figure B shows the inoculation results. Xe Statistical graph of pathogens after ΔXopQ mock (85-10). In the mock control group, stir1 stir2 The pathogen proliferation in the two double mutant lines was not significantly different from that in the WT plants. After flg22 pretreatment, the pathogen proliferation in the leaves of WT plants decreased significantly, indicating that flg22 pretreatment successfully triggered the PTI immune function in WT plants. stir1 stir2 After pretreatment with flg22, the pathogen proliferation of double mutant plants was not significantly reduced, and the pathogen proliferation in the flg22 pretreated double mutant group was significantly higher than that in the flg22 pretreated WT group (P<0.01).

[0080] 2.2 Regulatory effect on PTI immunosensitization function of transient expression materials Four-week-old *Nicotiana benthamiana* rootstock (WT) were obtained using the plant culture conditions described in Example 3. The pathogen culture method described in Example 3 was used to obtain transient transfections of overexpression empty vector, overexpression transient transfections, and point mutation transient transfections, respectively. Pst DC3000 ΔHopQ1 bacterial culture and Xe 85-10 ΔXopQ bacterial culture was used to prepare flg22 working solution with a working concentration of 1 μM.

[0081] Overexpression empty vector transient transfection solution, overexpression transient transfection solution, and point mutation transient transfection solution were inoculated onto whole leaves of WT plants, with 20 leaves inoculated for each type. The plants were then incubated in the dark at 25 °C for 1 day. After confirming that the vectors of the three transient transfection solutions were expressed in the plant leaves, an equal volume of flg22 working solution was injected according to the same method as in Example 4, 2.1, and the plants were incubated under normal conditions for 1 day. After the incubation period, an equal volume of... Pst DC3000 ΔHopQ1 bacterial solution or Xe 85-10 ΔXopQ bacterial suspension was used for inoculation, and the plants were then cultured under the original culture conditions using standard methods. Six biological replicates were set up to ensure the reliability of the experimental data. The number of pathogens was counted using the same counting method as in 2.1 of Example 4.

[0082] refer to Figure 8 As shown in Figure A, which depicts the vaccination process. Pst Pathogen statistics after DC3000 ΔHopQ1, Figure B shows the inoculation results. Xe 85-10 Statistical graph of pathogens after ΔXopQmock, GFP represents the transient transfection group overexpressing empty vector (control group), STIR1 represents the transient transfection group overexpressing empty vector, STIR1 E109A The term represents the point mutation transient conversion group. There was no significant difference in the number of pathogens between the point mutation cis-conversion group and the control group. However, the number of pathogens in the overexpression transient conversion group was significantly lower than both the point mutation cis-conversion group and the control group. This indicates that transient conversion of point mutation materials has no significant effect on the expression of STIR1 / 2 and the immune sensitization function of plants, while transient conversion of overexpression materials significantly enhances the immune sensitization function of plants.

[0083] The above results indicate that flg22-triggered immunosensitization depends on STIR1 / 2. When STIR1 / 2 is absent or undergoes point mutations that completely or partially disable its function, plants cannot effectively activate immunosensitization to resist pathogen invasion. Overexpression of STIR1 / 2, however, promotes immunosensitization in plants. This confirms that STIR1 / 2 is a key positive regulator of plant model immune responses and immunosensitization.

[0084] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0085] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. The application of STIR protein in enhancing plant-based model-triggered immune function, characterized in that, The STIR protein includes at least one of the following: STIR1 protein or a functional analog thereof, STIR2 protein or a functional analog thereof.

2. The application according to claim 1, characterized in that, The plants mentioned include Arabidopsis thaliana, tomato, soybean, rice, and Nicotiana benthamiana.

3. The application according to claim 1, characterized in that, The STIR protein is derived from *Nicotiana benthamiana*.

4. The application according to claim 1 or 3, characterized in that, The STIR1 protein has an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence that is at least 75% identical to it. Optionally, the STIR2 protein has an amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence having at least 75% identity with it.

5. The application according to claim 1, characterized in that, The gene encoding the STIR1 protein has a nucleotide sequence as shown in SEQ ID NO: 3 or a nucleotide sequence that is at least 80% identical to it; Optionally, the gene encoding the STIR2 protein has a nucleotide sequence as shown in SEQ ID NO: 4 or a nucleotide sequence having at least 80% identity with it.

6. A plant-based model-triggered immune function activator, characterized in that, The activator includes at least one of the STIR1 protein or its functional analogue, the STIR2 protein or its functional analogue, the gene encoding the STIR1 protein, and the gene encoding the STIR2 protein; Optionally, the activator further comprises an agriculturally acceptable carrier.

7. The use of the activator according to claim 6 in the preparation of plant immune enhancers, characterized in that, The plant immune enhancer is used to enhance the plant's model-triggered immune function to prevent and control plant diseases.

8. A method for enhancing immune function triggered by plant models, characterized in that, include: Upregulate STIR protein expression or increase STIR protein activity in plants; The STIR protein includes at least one of the following: STIR1 protein or a functional analog thereof, STIR2 protein or a functional analog thereof.

9. The method according to claim 8, characterized in that, The STIR1 protein has an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence that is at least 75% identical to it. Optionally, the STIR2 protein has an amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence having at least 75% identity with it.

10. The method according to claim 8, characterized in that, The plants mentioned include Arabidopsis thaliana, tomato, soybean, rice, and Nicotiana benthamiana.

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