Use of NIB1 gene in disease resisting and pest resisting of plants

Abstract

Provided is the use of the NIB1 gene in disease resisting and pest resisting of plants. Particularly provided is the use of the NIB1 gene or a coding polypeptide or accelerant thereof in enhancing the activity of NVR1 in plants, recruiting EDS1 to cell nucleuses or up-regulating disease resistance and pest resistance of plants. By means of up-regulating the expression level of NIB1, the present invention enhances disease resistance and pest resistance of plants.

Description

Application of NIB1 gene in plant disease and insect resistance Technical Field

[0001] The present invention relates to the fields of biotechnology and botany, and more particularly to the function of a rice RNA binding protein in disease and insect resistance and its application. Background Art

[0002] In organisms, RNA-binding proteins (RBPs) act as important post-transcriptional regulators, regulating RNA metabolism by binding to RNA. Traditional glycine-rich RNA-binding proteins (GR-RBPs) contain an RNA recognition motif (RRM) or cold-shock domain at their N-termini and a glycine-rich domain at their C-termini. These proteins participate in RNA splicing, transport, and editing, playing crucial roles in plant responses to cold, ultraviolet radiation, salinity, and pathogen infection (Ma et al., 2021a; Sachetto-Martins et al., 2000). This class of proteins constitutes the GRP IV family within the GRP superfamily (Ciuzan et al., 2015). In plant-pathogen interactions, the molecular mechanisms by which GR-RBPs recognize pathogen effectors and interact with NLR receptors have been extensively studied. The Pseudomonas syringae type III effector HopU1 can target Arabidopsis AtGRP7 and modify its RNA binding ability by ribosylating the arginine 49 of the RRM in AtGRP7, thereby weakening its role in promoting downstream defense responses and inhibiting plant immune responses (Fu et al., 2007; Jeong et al., 2011). CaGRP1 in pepper (Capsicum annuum) negatively regulates CaPIK1 (receptor-like cytoplasmic protein kinase 1) expression through its RRM domain, inhibiting CaPIK1-induced ROS production and cell death. CaGRP1-silencing plants upregulates the expression of CaPIK1, CaPR1, and CaDEF1, thereby enhancing pepper resistance to Xanthomonas campestris pv. Vesicatoria (Xcv) (Kim et al., 2015). In Nicotiana benthamiana, NbGRP7 interacts with the CNL-like receptor Gpa2, enhancing resistance to the potato white nematode (Globodera pallida). Furthermore, its RRM domain maintains Rx1 (the PVX immune receptor) homeostasis, implicating it in plant immunity to PVX infection (Sukarta et al., 2022). Research on how plant RNA-binding proteins recognize insect secreted nucleases and their catalytic products in pest and disease resistance has yet to be reported. Summary of the Invention

[0003] The inventors have provided for the first time a method for regulating the plant's defense response ability by regulating the expression level of the NIB1 gene in grass plants, thereby regulating the plant's disease and insect resistance.

[0004] In a first aspect, the present invention provides the use of the NIB1 gene or its encoded polypeptide or promoter in enhancing the activity of NVR1 in plants, recruiting EDS1 to the nucleus, or upregulating the plant's disease and insect resistance.

[0005] In one or more embodiments, the disease and insect resistance is selected from one or more of the following: resistance to rice leaf roller, resistance to cotton bollworm, and resistance to rice blast fungus.

[0006] In one or more embodiments, the promoter is selected from the group consisting of a small molecule compound, a nucleic acid molecule, or a combination thereof.

[0007] In one or more embodiments, the plant is a grass.

[0008] In one or more embodiments, the grass plant is rice, barley, wheat, oats, rye. Preferably, the grass plant is rice.

[0009] In one or more embodiments, the NIB1 gene is from a grass plant, preferably from rice.

[0010] In one or more embodiments, the polypeptide encoded by the NIB1 gene is selected from the group consisting of:

[0011] (a) a polypeptide having the sequence shown in SEQ ID NO: 2;

[0012] (b) a polypeptide derived from (a) formed by substituting, deleting or adding one or more (e.g., 1-20; preferably 1-10; more preferably 1-5) amino acid residues of the sequence shown in SEQ ID NO: 2, and having the function of the polypeptide of (a); or

[0013] (c) A polypeptide derived from (a) that has 90% (preferably 93%; more preferably 95% or 98%) or more homology with the polypeptide sequence of (a) and has the function of the polypeptide of (a).

[0014] In one or more embodiments, the nucleic acid sequence of the NIB1 gene is selected from:

[0015] (1) a polynucleotide sequence as shown in SEQ ID NO: 1 or a polynucleotide sequence having 80% (preferably 90%; more preferably 95% or 98%) or more homology thereto;

[0016] (2) a polynucleotide sequence having 1 to 60, preferably 1 to 30, more preferably 1 to 10 nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide sequence shown in SEQ ID NO: 1;

[0017] (3) A polynucleotide sequence complementary to the polynucleotide sequence described in any one of (1) to (2).

[0018] The present invention also provides a method for inactivating the NIB1 protein, comprising mutating the amino acid at position 49 and / or position 50. The NIB1 protein has the sequence shown in SEQ ID NO: 2.

[0019] In one or more embodiments, the mutation is a substitution or deletion mutation.

[0020] In one or more embodiments, the R at position 49 is mutated to A.

[0021] In one or more embodiments, the G at position 50 is mutated to an A.

[0022] A second aspect of the present invention provides a method for enhancing NVR1 activity in plants, recruiting EDS1 to the nucleus, and improving plant disease and insect resistance. The method comprises the steps of upregulating the expression or activity of the NIB1 gene in the plant. Preferably, the disease and insect resistance is selected from one or more of the following: resistance to rice leaf roller, resistance to cotton bollworm, and resistance to rice blast fungus.

[0023] In one or more embodiments, the plant is a grass.

[0024] In one or more embodiments, the grass plant is rice, barley, wheat, oats, rye. Preferably, the grass plant is rice.

[0025] In one or more embodiments, the polypeptide encoded by the NIB1 gene is selected from the group consisting of:

[0026] (a) a polypeptide having the sequence shown in SEQ ID NO: 2;

[0027] (b) a polypeptide derived from (a) formed by substituting, deleting or adding one or more (e.g., 1-20; preferably 1-10; more preferably 1-5) amino acid residues of the sequence shown in SEQ ID NO: 2, and having the function of the polypeptide of (a); or

[0028] (c) A polypeptide derived from (a) that has 90% (preferably 93%; more preferably 95% or 98%) or more homology with the polypeptide sequence of (a) and has the function of the polypeptide of (a).

[0029] In one or more embodiments, the nucleic acid sequence of the NIB1 gene is selected from:

[0030] (1) a polynucleotide sequence as shown in SEQ ID NO: 1 or a polynucleotide sequence having 80% (preferably 90%; more preferably 95% or 98%) or more homology thereto;

[0031] (2) a polynucleotide sequence having 1 to 60, preferably 1 to 30, more preferably 1 to 10 nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide sequence shown in SEQ ID NO: 1;

[0032] (3) A polynucleotide sequence complementary to the polynucleotide sequence described in any one of (1) to (2).

[0033] In one or more embodiments, the step of upregulating the expression or activity of the NIB1 gene in the plant comprises: transferring the NIB1 gene into the plant to obtain a transformed plant.

[0034] In one or more embodiments, the step of upregulating the expression or activity of the NIB1 gene in the plant comprises:

[0035] (1) Plant cells, tissues or organs are contacted with Agrobacterium containing a nucleic acid construct of the NIB1 gene, thereby transferring the nucleic acid construct into the plant cells, tissues or organs.

[0036] In one or more embodiments, the nucleic acid construct is an expression vector or a recombinant vector.

[0037] In one or more embodiments, the method of upregulating the expression of the NIB1 gene in a plant further comprises:

[0038] (2) selecting plant cells, tissues, organs or seeds into which the NIB1 gene has been transferred; and

[0039] (3) Regenerating the plant cells, tissues, organs or seeds in step (3) into plants.

[0040] The present invention also provides the use of the NIB1 gene as a target in screening potential agents, wherein the potential agents can increase the activity of NVR1 in plants, recruit EDS1 to the cell nucleus, and enhance the plant's disease and insect resistance.

[0041] In one or more embodiments, the screening comprises the steps of: (1) contacting a candidate agent with a NIB1 protein or a nucleic acid molecule encoding the NIB1 protein or a system containing the NIB1 protein, and (2) detecting changes in the expression or activity of the protein or the nucleic acid molecule encoding the NIB1 protein, which can indicate that the candidate agent is a target potential agent. If the candidate agent can increase the expression or activity of the NIB1 protein or the nucleic acid molecule encoding the NIB1 protein, it indicates that the candidate agent is a potential agent for increasing the activity of NVR1 in plants, recruiting EDS1 to the nucleus, and enhancing the plant's resistance to pests and diseases. In a preferred embodiment, step (1) comprises: in a test group, adding the candidate agent to a system containing the NIB1 protein or the nucleic acid molecule encoding the NIB1 protein, and step (2) comprises: detecting the expression or activity of the NIB1 protein in the system of the test group and comparing it with a control group, wherein the control group is the same system without the addition of the candidate agent. If the expression of NIB1 protein in the test group is statistically higher (preferably significantly higher, such as 20% or more higher, preferably 50% or more higher, and more preferably 80% or more higher) than that in the control group, it indicates that the candidate is a potential agent for increasing the activity of NVR1 in plants, recruiting EDS1 to the cell nucleus, and enhancing the plant's resistance to diseases and insect pests.

[0042] In one or more embodiments, the screening comprises the steps of: (1) contacting a candidate agent, NIB1 protein or a nucleic acid molecule encoding thereof with (a) NVR1 protein or a nucleic acid molecule encoding thereof and / or (b) EDS1 protein or a nucleic acid molecule encoding thereof, or (c) a system containing (a) and / or (b), and (2) if any one or more of the following conditions are detected, indicating that the candidate agent is a potential agent for increasing the activity of NVR1 in plants, recruiting EDS1 to the cell nucleus, and enhancing the disease and insect resistance of plants: (A) enhancing the interaction between NIB1 protein and NVR1 protein, (B) enhancing the activity of NVR1 protein, (C) enhancing the interaction between NIB1 protein and EDS1 protein, (D) enhancing the effect of NIB1 in recruiting EDS1 to condensates.

[0043] In one or more embodiments, the screening comprises the steps of: (1) adding a candidate agent to a system containing NIB1 protein or a nucleic acid molecule encoding it and NVR1 protein or a nucleic acid molecule encoding it in a test group; and (2) detecting the activity of the NVR1 protein or the interaction between the NIB1 protein and the NVR1 protein in the test group system and comparing it with a control group, wherein the control group is the same system without the addition of the candidate agent. If the activity of the NVR1 protein or the interaction between the NIB1 protein and the NVR1 protein in the test group is statistically higher (preferably significantly higher, such as higher by 20% or more, preferably higher by 50% or more, and more preferably higher by 80% or more) than that in the control group, it indicates that the candidate agent is a potential agent for increasing the activity of NVR1 in plants, recruiting EDS1 to the nucleus, and enhancing the plant's resistance to diseases and insects.

[0044] In one or more embodiments, the screening comprises the steps of: (1) adding a candidate agent to a system containing NIB1 protein or a nucleic acid molecule encoding it and EDS1 protein or a nucleic acid molecule encoding it in a test group; and (2) detecting the interaction between the NIB1 protein and the EDS1 protein or the EDS1 protein in the aggregates in the test group, and comparing the results with those in a control group, wherein the control group is the same system without the candidate agent. If the interaction between the NIB1 protein and the EDS1 protein or the EDS1 protein content in the aggregates in the test group is statistically higher (preferably significantly higher, such as by 20% or more, preferably by 50% or more, and more preferably by 80% or more) than that in the control group, the candidate agent is a potential agent for increasing NVR1 activity in plants, recruiting EDS1 to the nucleus, and enhancing plant resistance to pests and diseases.

[0045] In one or more embodiments, the system is selected from: a cell system (such as a cell expressing NIB1 protein) (or a cell culture system), a subcellular system, a solution system, a tissue system, an organ system, or an animal system.

[0046] In one or more embodiments, the interaction between proteins is detected by MST assay, Co-IP or bimolecular luciferase complementation assay.

[0047] In one or more embodiments, protein expression is detected by detecting antibody-antigen reaction, detecting mRNA content, such as Western, ELISA or Southern.

[0048] In one or more embodiments, the EDS1 protein content in the aggregates is detected by co-localization observation under a microscope. For example, NIB1 and EDS1 are linked to vectors with different fluorescent markers, respectively, and transformed into tobacco leaves for co-localization observation under a microscope. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1: Interaction between NIB1 and NVR1.

[0050] A, MST experiments showed that NIB1 directly binds to NVR1, with NIB1 acting as a ligand.

[0051] B, Ex vivo pull-down assay confirmed the interaction between NIB1 and NVR1.

[0052] C, Co-IP verified the interaction between NVR1 and NIB1.

[0053] D, Schematic diagram of the NIB1 protein structure. RNA Recognition Motif; Glycine-rich region.

[0054] E, Bimolecular luciferase complementation assay in tobacco confirmed that the interaction between NIB1 and NVR1 depends on the RRM domain.

[0055] Figure 2: Amino acid residues 49 and 50 of NIB1 are very important for the function of NIB1.

[0056] A, Schematic diagram of NIB1 results, with arginine at position 49 and glycine at position 50.

[0057] B, Molecular docking results showed that NIB1 can bind to 2',3'-cAMP, and the amino acids responsible for recognition are R49 and G50.

[0058] C, the amino acid that binds to 2',3'-cGMP is G50.

[0059] D, MST experiments show that NIB1 binds to 2', 3'-cAMP and NIB1 R49A Reduced binding capacity and NIB1 G50A Loss of binding ability.

[0060] E, Fluorescence confocal microscopy showed that in the presence of NVR1, NIB1-YFP cells could form condensates in the nucleus. R49 With NIB1 G50A Then you can't.

[0061] F, Fluorescence confocal microscopy showed that 2',3'-cAMP promoted the formation of NIB1-YFP condensates in the nucleus. R49 With NIB1 G50A Loss of this capability. Scale bars are all 5 microns.

[0062] G, Transient expression of NIB1-YFP in tobacco leaves can cause plant cell death. R49A With NIB1 G50A Does not cause plant cell necrosis.

[0063] H, Feeding experiments with cotton bollworms showed that transiently expressed NIB1 positively regulated cotton bollworm resistance.

[0064] Figure 3: NIB1 accelerates NVR1 activity.

[0065] A, Urea SDS-PAGE detection of NIB1-YFP and NIB1 ΔIDR Effects on the catalytic activity of NVR1.

[0066] BC, Characterization of NIBI-YFP and NIB1 by detecting 2',3'-cAMP / cGMP by HPLC-MS ΔIDREffects on the catalytic activity of NVR1.

[0067] D, Using Agrobacterium-mediated tobacco transient expression system, NVR1-HA+YFP, NVR1-HA+NIB1-YFP and NVR1-HA+NIB1 were expressed in tobacco leaves. ΔIDR -YFP. Necrosis was observed and photographed 3 days later. Scale bar represents 1 cm.

[0068] EH quantified AD respectively.

[0069] IJ, Characterization of NIB1-YFP and NIB1 by detecting 2',3'-cAMP / cGMP using HPLC-MS R49A With NIB1 G50A Effects on the catalytic activity of NVR1.

[0070] KL, used the Agrobacterium-mediated tobacco transient transformation system to express NVR1+NIB1, NVR1+NIB1, and NVR1+NIB1. R49A and NVR1+NIB1 G50A , three days later, samples were taken and photographs were taken.

[0071] Figure 4: NIB1 can recruit EDS1 to condensates.

[0072] A, Bimolecular luciferase complementation assay confirms the interaction between NIB1 and EDS1. The red-blue gradient indicates the strength of the interaction.

[0073] B, Semi-in vitro pull-down assay confirms the interaction between His-NIB1 and EDS1-Flag. Immunoblotting was performed using His and Flag antibodies, respectively.

[0074] C, Interaction between NIB1-Flag and EDS1-GFP was confirmed by immunoprecipitation in tobacco leaves. Flag and GFP antibodies were used for hybridization, respectively.

[0075] D, Bimolecular fluorescence complementation (BiFC) was used to verify the interaction between EDS1 and NIB1 in tobacco leaves. Scale bar: 20 μm.

[0076] E, Colocalization results show that NIB1 recruits EDS1 to condensates. Scale bar: 5 μm.

[0077] Figure 5: NIB1 positively regulates plant disease and insect resistance.

[0078] A, Disease development in ZH11, NIB1-KO, and NIB1-OE plants 5 days after inoculation with rice blast fungus (TH12).

[0079] B, Lesion length 5 days after infection with rice blast fungus. A total of 11 leaves were counted.

[0080] C, qRT-PCR was performed to calculate the relative amount of rice blast fungus using the rice internal reference gene ACTIN and the pathogen 28S rDNA.

[0081] D, Insect resistance of ZH11, NIB1-KO, and NIB1-OE plants 5 days after inoculation with rice leaf folder.

[0082] E, Rice leaf folder after three days of feeding.

[0083] F, Weighing of rice leaf folder. DETAILED DESCRIPTION

[0084] The inventors revealed for the first time that by targeted regulation of the expression level of the NIB1 gene in grass plants, the ability of the plant's defense response can be significantly adjusted, thereby regulating the plant's disease and insect resistance, including resistance to rice leaf roller, cotton bollworm and rice blast fungus.

[0085] As used herein, "grasses" are rice, barley, wheat, oats, rye.

[0086] In the present invention, the term "NIB1" refers to a polypeptide having NIB1 activity, as defined in SEQ ID NO: 2, or a polypeptide encoded by the NIB1 gene with the ID LOC_Os03g46770. The term also encompasses variants of SEQ ID NO: 2 that possess the same function as NIB1. These variants include (but are not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 1-50, preferably 1-30, 1-20, 1-10, 1-8, or 1-5), as well as additions or deletions of one or more amino acids (typically within 20, preferably within 10, and more preferably within 5) at the C-terminus and / or N-terminus. For example, substitutions with amino acids having similar or similar properties generally do not alter protein function. In the art, amino acids with similar properties often refer to families of amino acids with similar side chains, which are well-defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, lactic acid, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For another example, adding one or more amino acids to the amino and / or carboxyl termini generally does not alter the function of a polypeptide or protein. Conservative amino acid substitutions for many common, known non-genetically encoded amino acids are known in the art. Conservative substitutions for other non-encoded amino acids can be determined based on a comparison of their physical properties with the properties of the genetically encoded amino acids.

[0087] The variant forms of polypeptides include: homologous sequences, conservative variants, allelic variants, natural mutants, and induced mutants.

[0088] Any polypeptide with high homology to NIB1 (e.g., 70% or greater homology to the sequence set forth in SEQ ID NO: 2; preferably, 80% or greater homology; more preferably, 90% or greater homology, such as 95%, 98%, or 99% homology) and having similar or identical functions to NIB1 is also encompassed by the present invention. The "same or similar functions" primarily refer to regulating disease and pest resistance in crops (e.g., rice).

[0089] The present invention also includes analogs of the polypeptides claimed for protection. These analogs may differ from the natural SEQ ID NO: 2 in terms of amino acid sequence, or in terms of modifications that do not affect the sequence, or both. Analogs of these proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, or by site-directed mutagenesis or other known biological techniques. Analogs also include analogs with residues other than natural L-amino acids (such as D-amino acids), as well as analogs with non-natural or synthetic amino acids (such as β, γ-amino acids). It should be understood that the proteins of the present invention are not limited to the representative proteins exemplified above.

[0090] Modifications (which generally do not alter the primary structure) include chemical derivatization of proteins in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those that occur during protein synthesis and processing. Such modifications can be accomplished by exposing the protein to glycosylation enzymes (e.g., mammalian glycosylases or deglycosylases). Modifications also include sequences containing phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, and phosphothreonine).

[0091] The polypeptide fragments, derivatives or analogs of the present invention can be: (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having a substituent group in one or more amino acid residues; or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein). According to the definition herein, these fragments, derivatives and analogs fall within the scope well known to those skilled in the art.

[0092] Furthermore, any biologically active fragment of NIB1 can be used in the present invention. Herein, a biologically active fragment of NIB1 refers to a polypeptide that retains all or part of the function of full-length NIB1. Typically, the biologically active fragment retains at least 50% of the activity of full-length NIB1. More preferably, the biologically active fragment retains 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of full-length NIB1.

[0093] The present invention also relates to polynucleotide sequences encoding NIB1 of the present invention or variants, analogs, or derivatives thereof. The polynucleotide may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to the coding region sequence set forth in SEQ ID NO: 1 or a degenerate variant.

[0094] The present invention also relates to variants of the above-mentioned polynucleotides, which encode fragments, analogs, and derivatives of polypeptides having the same amino acid sequence as the present invention. Variants of such polynucleotides may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes. As used herein, a degenerate variant in the present invention refers to a nucleic acid sequence that encodes a protein having SEQ ID NO: 2 but differs from the coding region sequence shown in SEQ ID NO: 1. A "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide or a polynucleotide further including additional coding and / or non-coding sequences.

[0095] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.

[0096] It should be understood that although the gene provided in the examples of the present invention is derived from rice, NIB1 gene sequences derived from other similar plants (especially plants belonging to the same family or genus as rice) that have a certain degree of homology (e.g., greater than 70%, such as 80%, 85%, 90%, 95%, or even 98% sequence identity) with the sequence of the present invention (preferably, the sequence shown in SEQ ID NO: 1) are also included within the scope of the present invention, as long as those skilled in the art can easily isolate the sequence from other plants based on the information provided in this application after reading this application. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0097] The full-length NIB1 nucleotide sequence or fragments thereof of the present invention can generally be obtained by PCR amplification, recombinant methods, or synthetic methods. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly the open reading frame sequences, and amplified using a commercially available DNA library or a cDNA library prepared by conventional methods known to those skilled in the art as a template to obtain the relevant sequence. Long sequences often require two or more PCR amplifications, followed by splicing the fragments amplified from each amplification in the correct order. Once the relevant sequence is obtained, recombinant methods can be used to obtain it in large quantities. Typically, the sequence is cloned into a vector, transferred into cells, and then isolated from the propagated host cells by conventional methods.

[0098] In addition, artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then connecting them, very long fragments of sequence can be obtained. At present, DNA sequences encoding proteins of the present invention (or their fragments, or their derivatives) can be obtained completely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0099] The present invention also provides a nucleic acid construct comprising the gene of the present invention. As a preferred embodiment, the promoter downstream of the nucleic acid construct comprises a multiple cloning site or at least one restriction enzyme cleavage site. When it is necessary to express the target gene of the present invention, the target gene is connected to a suitable multiple cloning site or restriction enzyme cleavage site, thereby operably connecting the target gene to the promoter. As another preferred embodiment, the nucleic acid construct comprises (from 5' to 3' direction): a promoter, a target gene, and a terminator. If necessary, the nucleic acid construct may further comprise an element selected from the following groups: a 3' polynucleotide signal; a non-translated nucleic acid sequence; a transport and targeting nucleic acid sequence; a resistance selection marker (dihydrofolate reductase, neomycin resistance, hygromycin resistance, and green fluorescent protein, etc.); an enhancer; or an operator.

[0100] Methods for preparing nucleic acid constructs are well known to those of ordinary skill in the art. Expression vectors can be bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors. In short, any plasmid or vector can be used as long as it can replicate and be stable in the host.

[0101] Those skilled in the art can construct expression vectors containing the genes of the present invention using well-known methods. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. When constructing recombinant expression vectors using the genes of the present invention, any enhancing, constitutive, tissue-specific, or inducible promoter can be added before the transcription initiation nucleotide.

[0102] Genes or nucleic acid constructs comprising the present invention can be used to transform appropriate host cells to express proteins. Host cells can be prokaryotes, such as Escherichia coli, Streptomyces, or Agrobacterium; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Those skilled in the art will appreciate how to select appropriate vectors and host cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques familiar to those skilled in the art. When the host is a prokaryotic organism (such as Escherichia coli), CaCl treatment or electroporation can be used. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.). Plants can also be transformed using methods such as Agrobacterium transformation or gene gun transformation, such as the leaf disc method, immature embryo transformation method, and flower bud immersion method. Transformed plant cells, tissues, or organs can be regenerated into plants using conventional methods to obtain transgenic plants. When the polynucleotide is expressed in higher eukaryotic cells, transcription can be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting DNA elements, usually about 10 to 300 base pairs long, that act on promoters to increase gene transcription.

[0103] It is clear to those skilled in the art how to select appropriate vectors, promoters, enhancers and host cells.

[0104] The polypeptides described herein can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include (but are not limited to): conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic shock, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0105] Transformation of hosts with recombinant DNA can be performed using conventional techniques familiar to those skilled in the art. Plants can be transformed using methods such as Agrobacterium transformation or gene gun transformation, for example, spraying, leaf disc, and rice embryo transformation. Transformed plant tissues or organs can be regenerated into plants using conventional methods to obtain plants with altered traits.

[0106] The present invention provides uses of the NIB1 gene for regulating NVR1 activity in plants, recruiting EDS1 to the nucleus, or regulating plant disease and pest resistance. As a preferred embodiment, the NIB1 gene can be used to enhance NVR1 activity in plants and increase plant disease and pest resistance.

[0107] The present invention also relates to a NIB1 promoter and its use. Since the NIB1 promoter can increase the expression and / or activity of NIB1, the promoter can regulate the activity of NVR1 in plants and regulate the plant defense response by affecting NIB1, thereby achieving the plant's ability to resist diseases and insects.

[0108] In one aspect, any substance that can enhance NIB1 activity, improve its stability, promote its expression, prolong its effective duration, or promote its gene transcription and translation can be used in the present invention as a "promoter" of the NIB1 gene to regulate plant disease and pest resistance. For example, expression vectors that enhance the transcription, expression, or activity of the NIB1 gene can be used.

[0109] The present invention also provides a method for regulating NVR1 activity in plants, recruiting EDS1 to the cell nucleus, or regulating plant disease and insect resistance. The method comprises upregulating the expression or activity of the NIB1 gene in a plant, thereby regulating NVR1 activity and regulating plant disease and insect resistance. Once the use of the NIB1 gene is known, various methods well known in the art can be used to upregulate NIB1 gene expression. For example, an expression unit carrying the NIB1 gene (such as an expression vector or virus) can be delivered to a target site using methods known in the art to induce expression of active NIB1.

[0110] In one embodiment of the present invention, the NIB1 gene is cloned into an appropriate vector using conventional methods, and the nucleic acid construct containing the NIB1 gene is introduced into plant tissues or organs to cause the plant to express the NIB1 gene. Plants overexpressing the NIB1 gene can be obtained by regenerating the plant tissues or organs into plants.

[0111] In one or more embodiments, the method for upregulating the expression of the NIB1 gene comprises:

[0112] (1) providing Agrobacterium carrying a nucleic acid construct containing the NIB1 gene,

[0113] (2) contacting plant cells, tissues or organs with the Agrobacterium in step (1), thereby transferring the nucleic acid construct into the plant tissues or organs.

[0114] In one or more embodiments, the method of upregulating the expression of the NIB1 gene in a plant further comprises:

[0115] (3) selecting plant tissues, organs or seeds into which the NIB1 gene has been transferred; and

[0116] (4) Regenerating the plant tissue, organ or seed in step (3) into a plant.

[0117] The present invention also provides the use of the NIB1 gene as a target in screening potential agents, wherein the potential agents can increase the activity of NVR1 in plants, recruit EDS1 to the cell nucleus, and enhance the plant's disease and insect resistance.

[0118] Herein, the screening comprises the steps of: (1) contacting a candidate agent with a NIB1 protein or its encoding nucleic acid molecule, or a system containing the same, and (2) detecting changes in the expression or activity of the protein or its encoding nucleic acid molecule, which can indicate that the candidate agent is a target potential agent. If the candidate agent can increase the expression or activity of the NIB1 protein or its encoding nucleic acid molecule, it indicates that the candidate agent is a potential agent for increasing the activity of NVR1 in plants, recruiting EDS1 to the cell nucleus, and enhancing the plant's resistance to pests and diseases. In a preferred embodiment, step (1) comprises: in a test group, adding the candidate agent to a system containing the NIB1 protein or its encoding nucleic acid molecule, and step (2) comprises: detecting the expression or activity of the NIB1 protein in the test group system and comparing it with a control group, wherein the control group is the same system without the addition of the candidate agent. If the expression of NIB1 protein in the test group is statistically higher (preferably significantly higher, such as 20% or more higher, preferably 50% or more higher, and more preferably 80% or more higher) than that in the control group, it indicates that the candidate is a potential agent for increasing the activity of NVR1 in plants, recruiting EDS1 to the cell nucleus, and enhancing the plant's resistance to diseases and insect pests.

[0119] In one or more embodiments, the screening comprises the steps of: (1) contacting a candidate agent, NIB1 protein or a nucleic acid molecule encoding thereof with (a) NVR1 protein or a nucleic acid molecule encoding thereof and / or (b) EDS1 protein or a nucleic acid molecule encoding thereof, or (c) a system containing (a) and / or (b), and (2) if any one or more of the following conditions are detected, indicating that the candidate agent is a potential agent for increasing the activity of NVR1 in plants, recruiting EDS1 to the cell nucleus, and enhancing the disease and insect resistance of plants: (A) enhancing the interaction between NIB1 protein and NVR1 protein, (B) enhancing the activity of NVR1 protein, (C) enhancing the interaction between NIB1 protein and EDS1 protein, (D) enhancing the effect of NIB1 in recruiting EDS1 to condensates.

[0120] In one or more embodiments, the screening comprises the steps of: (1) adding a candidate agent to a system containing NIB1 protein or a nucleic acid molecule encoding it and NVR1 protein or a nucleic acid molecule encoding it in a test group; and (2) detecting the activity of the NVR1 protein or the interaction between the NIB1 protein and the NVR1 protein in the test group system and comparing it with a control group, wherein the control group is the same system without the addition of the candidate agent. If the activity of the NVR1 protein or the interaction between the NIB1 protein and the NVR1 protein in the test group is statistically higher (preferably significantly higher, such as higher by 20% or more, preferably higher by 50% or more, and more preferably higher by 80% or more) than that in the control group, it indicates that the candidate agent is a potential agent for increasing the activity of NVR1 in plants, recruiting EDS1 to the nucleus, and enhancing the plant's resistance to diseases and insects.

[0121] In one or more embodiments, the screening comprises the steps of: (1) adding a candidate agent to a system containing NIB1 protein or a nucleic acid molecule encoding it and EDS1 protein or a nucleic acid molecule encoding it in a test group; and (2) detecting the interaction between the NIB1 protein and the EDS1 protein or the EDS1 protein in the aggregates in the test group, and comparing the results with those in a control group, wherein the control group is the same system without the candidate agent. If the interaction between the NIB1 protein and the EDS1 protein or the EDS1 protein content in the aggregates in the test group is statistically higher (preferably significantly higher, such as by 20% or more, preferably by 50% or more, and more preferably by 80% or more) than that in the control group, the candidate agent is a potential agent for increasing NVR1 activity in plants, recruiting EDS1 to the nucleus, and enhancing plant resistance to pests and diseases.

[0122] As used herein, "system" is selected from: a cell system (e.g., cells expressing NIB1 protein) (or a cell culture system), a subcellular system, a solution system, a tissue system, an organ system, or an animal system. In one or more embodiments, protein-protein interactions are detected by MST assays, Co-IP, or bimolecular luciferase complementation assays. In one or more embodiments, protein expression is detected by detecting antibody-antigen reactions or mRNA levels, such as Western blotting, ELISA, or Southern blotting. In one or more embodiments, EDS1 protein levels in aggregates are detected by microscopic colocalization observation. For example, NIB1 and EDS1 are separately linked to vectors with different fluorescent markers, transformed into tobacco leaves, and then colocalized under a microscope for observation.

[0123] Other aspects of the present invention will be apparent to those skilled in the art from the disclosure herein. The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. Experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0124] Example

[0125] Example 1, NIB1 interacts with NVR1

[0126] a: MST detection of the interaction between NIB1 and NVR1

[0127] Purified NIB1 protein was labeled with RED-NHS using a kit (RED-NHS Protein Labeling Kit (Amino Labeling - RED Channel, NT-L111) to obtain NHS-NIB1 protein. NHS-NIB1 was then tested for aggregation and capillary adsorption. If no adsorption or aggregation was observed, subsequent experiments could be performed. If adsorption and aggregation were observed, Tween could be added as needed, but the concentration should not be too high. Ligand (purified E. coli NVR1 protein) was diluted by first preparing 20 μL of protein (at a concentration 20-50 times the Kd) and adding 10 μL of PBS to each of 15 PCR tubes. The protein concentration was then diluted sequentially. NHS-NIB1 protein was then added to each of these PCR tubes and pipetted evenly. The sample was then adsorbed using a capillary tube for detection (Figure 1, A).

[0128] b: Ex vivo pull-down experiment

[0129] NIB1 was constructed in the pEAQ-flag vector (available in our laboratory). After successful sequencing, Agrobacterium was transformed: competent Agrobacterium GV3101 (Weidi Biotech) was thawed on ice until a mixture of ice and water was present. 1 μL of the plasmid containing the target fragment was added to the competent cells. The cells were placed on ice for 30 minutes, cooled in liquid nitrogen for 30 seconds, placed in a 37°C water bath for 5 minutes, and then placed on ice for 3 minutes. 100 μL of liquid LB medium was added to the competent cells and cultured in a 28°C incubator with shaking for 3 hours. 50 μL of the bacterial suspension was spread on solid culture medium of the corresponding resistance and cultured at 28°C for 48 hours. A single colony was selected as the successfully transformed Agrobacterium.

[0130] Transient transformation of tobacco: Pick a single clone the night before and transfer it to liquid RKG medium. Centrifuge the overnight culture at 5000 rpm for 5 minutes. Carefully pour out the supernatant liquid medium. Add 1 mL of resuspension solution to the precipitate and resuspend it with a pipette tip to obtain a uniform resuspension of the bacterial suspension. Use NanoDrop to measure OD 600nm The bacterial concentration was adjusted with resuspension solution, and the OD of single bacterial cell injection was 600nm The OD value of the two bacterial solutions was about 0.6. 600nm About 1, after mixing, ensure the OD of a single bacterium 600nm At around 0.5, use a syringe with a removed needle to inject the tobacco leaves and mark them.

[0131] Two days later, extract total tobacco leaf protein: Place 0.25g of tobacco leaves in a 2mL centrifuge tube containing steel balls, snap-freeze in liquid nitrogen, and completely pulverize the leaves in a crusher. Add 1mL of IP-lysis buffer to the tube, vortex for 1 minute, and ice-bathe for 5 minutes. Repeat 3N5 times. Centrifuge at 14,000 rpm for 10 minutes. Remove the supernatant and transfer it to a new centrifuge tube. Centrifuge at 14,000 rpm for 10 minutes. Repeat two to three times. The supernatant is the total tobacco protein solution, and the concentration is measured for subsequent experiments. At the same time, another His-tagged protein, NVR1, needs to be purified.

[0132] Transfer 50 μL of the nickel column bound to the His recombinant protein to a 1.5 mL centrifuge tube and add 800 μL of extracted tobacco total protein. Incubate with rotation at 4°C for 3 h. Centrifuge the protein solution at 2500 rpm at 4°C for 3 min, and carefully aspirate the supernatant. Add 800 μL of Co-IP buffer (50 mM Tris-HCl, 150 mM NaCl, 20% glycerol, 0.5% NP-40, 1× PIC (Full Gold)) to the centrifuge tube, rotate for 5 min, and centrifuge at 2500 rpm at 4°C for 3 min. Carefully aspirate the supernatant. Repeat five times, add 40 μL of 4× protein loading buffer (Tanon, 180-8210D), and aspirate 20 μL for western blotting using a Flag antibody (ZSGB-BIO, TA-05). At the same time, His recombinant protein and tobacco leaf protein bound to the nickel column were taken as input, and immunoblotting experiments were performed using His and Flag antibodies, respectively (Figure 1, B).

[0133] c: Co-IP verification of the interaction between NVR1 and NIB1 in vivo

[0134] The proteins to be verified for interaction, NVR1 and NIB1, were ligated into the pCAMBIA1300-YFP (preserved in the laboratory) and pEAQ-FLAG vectors, respectively. After transformation into Agrobacterium tumefaciens GV3101, the two bacterial cultures were mixed for transient transformation of tobacco leaves. Two days later, total protein was extracted from tobacco leaves, and 30 μL of GFP beads (Chromotek, gta-20) were added to the total protein. The mixture was incubated with rotation overnight at 4°C. The next day, the cells were centrifuged at 2500 rpm for 3 minutes. The supernatant was gently aspirated, and 500 μL of IP Lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 20% glycerol, 0.5% NP-40, 1× PIC (Full Gold)) was added. The cells were incubated with rotation for 5 minutes, followed by centrifugation at 2500 rpm for 3 minutes. This process was repeated five times. Protein interactions were detected by western blotting using a Flag antibody. Total protein was also probed using Flag and GFP antibodies (Figure 1, C).

[0135] d: Bimolecular luciferase complementation assay of NIB1 binding to NVR1

[0136] The proteins NVR1 and NIB1, used to verify their interaction, were ligated into the JW772-35S-cLUC vector (maintained in our laboratory) and the JW771-35S-nLUC vector (maintained in our laboratory), respectively. After sequencing, the two vectors were transformed into Agrobacterium tumefaciens GV3101. After two days of culture at 28°C on RKG solid medium (10 g tryptone, 5 g yeast extract, 10 g NaCl, 15 g agar, 10 g tryptone, 5 g yeast extract, 10 g NaCl, 15 g agar (solid), Thermo Scientific, supplemented with 25 mg / L kanamycin, 25 mg / L gentamycin, and 50 mg / L kanamycin, respectively), a single colony was picked and incubated in 4 mL RKG liquid medium (10 g tryptone, 5 g yeast extract, 10 g NaCl), cultured overnight at 28°C, and then transformed into tobacco leaves according to the tobacco transient transformation protocol. The leaves were then labeled. Two days later, luciferase substrate (D-Luciferin (potassium salt, APExBIO) was injected into the transient transformation site using a 1 mL needle-free syringe, and the leaves were placed in an exposure instrument for luminescence signal detection and pseudocolor addition ( Figure 1 , D).

[0137] Example 2: NIB1 amino acid residues 49 and 50 are very important for NIB1 to function.

[0138] a: Protein structure simulation and key amino acid site mutation

[0139] Using the crystal structure of tobacco RNA-binding protein NbGRP7 as a reference, molecular modeling of NIB1 was performed to obtain its possible protein structure and its binding sites for 2', 3'-cAMP and 2', 3'-cGMP, including amino acid residues 49 and 50 in the RRM domain (Figure 2, AC). Site-directed mutagenesis was performed on these sites (the mutation method was substitution mutation, segmenting the gene at the mutation site, introducing mutant nucleic acid bases in the primers, and constructing the vector using multi-segment homologous recombination), and microthermophoresis (MST) was used to detect its ability to bind small molecules. The results showed that NIB1 has the ability to bind to 2', 3'-cAMP, but NIB1 R49A With NIB1 G50A The binding ability was weakened or even disappeared (Figure 2, D).

[0140] b: Fluorescence confocal microscopy observation of NIB1 condensate formation in plant nuclei

[0141] Cut tobacco plant leaves of a certain size, place them upside down on a glass slide, add a drop of ddH2O, gently cover with a coverslip, and absorb excess water; place the slide upside down on the stage; first use a low-power lens to find the target field of view, and move to the center of the field of view, switch to a high-power lens, select the corresponding fluorescence excitation wavelength and receiving wavelength, adjust the excitation intensity and related parameters, adjust the Z axis, and collect images. During the shooting process, it should be ensured that there is no overexposure; the YFP used in this study was collected through the GFP channel, with an excitation wavelength of 488nm and a receiving wavelength of 498-550nm; the collected images were added with a ruler and quantified using Image J software. NIB1-YFP tobacco leaves were observed under a microscope. NIB1-YFP can form bright dots in the cell nucleus, but NIB1 R49A With NIB1 G50A It cannot (Figure 2, EF).

[0142] c: Tobacco expressed protein

[0143] Transform the constructed vector into Agrobacterium GV3101 competent cells, and incubate the plates upside down at 28°C for 2 days. Two days later, pick the single clones on the plates and transfer them to 4mL RKG liquid culture medium, incubate them on a shaker at 28°C overnight, amplify the small amount of cultured Agrobacterium at 1:100 into fresh RKG medium, and incubate them on a shaker at 28°C overnight. Centrifuge at 4°C, 5000g, for 5 minutes, discard the excess LB, and resuspend the bacteria with tobacco instantaneous resuspension solution to make the bacterial solution OD600 of 0.6. Let it stand at room temperature for 2-3 hours and prepare for injection. Separately transfect NIB1-YFP and NIB1 R49A With NIB1 G50A Two days after the injection of tobacco, the results showed that the site of NIB1-YFP injection could cause plant cell death, but NIB1 R49A With NIB1 G50A It cannot (Figure 2, G).

[0144] d: Cotton bollworm feeding experiment

[0145] Large-scale injection of NIB1-YFP, NIB1 R49A With NIB1 G50A Afterwards, tobacco leaves were cut into the same size, and leaves of the same size and position were placed in a 6-well culture dish. Second-instar cotton bollworms of the same size (purchased from Henan Keyun Biological Company) were picked and placed in the culture dish and weighed. The weight was weighed every other day, and fresh plant leaves of the same size were added every other day. After feeding for three days, the weight was weighed again. The data obtained was subtracted from the data on the first day of feeding to obtain the weight gain of the cotton bollworm. The results showed that NIB1 improved the ability of tobacco to resist cotton bollworms. NIB1 R49A With NIB1 G50AIt cannot (Figure 2, H).

[0146] Agrobacterium infection buffer: 10 mM MgCl2, 10 mM MES, 150 μM acetosyringone, adjusted to pH 5.8 with KOH.

[0147] Example 3: NIB1 Accelerates NVR1 Activity

[0148] a: In vitro detection of the effect of NIB1 on NVR1 activity

[0149] In vitro transcription was performed using DNA as a template and Cy5-UTP as a substrate. Cy5-labeled RNA was obtained after removing the DNA template. The reaction was then carried out at 25°C for 25 min using a mixture of NVR1 and substrate at a ratio of 1:100 and NIB1:NVR1 at a ratio of 1:1. The enzyme activity was terminated by adding a high salt buffer. The enzyme was purified using phenol-chloroform and isopropanol. After redissolution, 2×TBE urea loading buffer (0.5×TBE, 6 M urea, 0.01% bromophenol blue, 15% PM 400), reacted at 95℃ for 3 min, and immediately placed on ice for urea polyacrylamide gel electrophoresis. Signals were scanned and detected using TYPHOON5, and bands were quantified using Image J. As shown in Figure 3, A and E, NIB1 can significantly promote the catalytic activity of NVR1, but NIB1 ΔIDR Then you can't.

[0150] b: Detection of catalytic products by high performance liquid chromatography

[0151] Small molecules were separated using an XSelect HSS T3 XP column (Waters). The aqueous phase consisted of water containing 2 mM ammonium acetate, and the organic phase consisted of methanol containing 2 mM ammonium acetate. Separation conditions were as follows:

[0152] The results shown in Figure 3 B, C, F, G, I and J show that NIB1 can promote the production of more active small molecules 2', 3'-cAMP and 2', 3'-cGMP, but NIB1 ΔIDR With NIB1 R49A With NIB1 G50A Does not have this ability.

[0153] c: Co-transformation in tobacco cells to verify the effect of NIB1 on NVR1-induced necrosis

[0154] The constructed vectors NVR1-YFP and NIB1-YFP (YFP) were co-transformed into Agrobacterium GV3101 competent cells and the plates were incubated upside down at 28°C for 2 days. Two days later, single colonies were picked from the plates and transferred to 4 mL of RKG liquid medium and cultured overnight at 28°C in a shaker. The small amount of Agrobacterium culture was amplified 1:100 into fresh RKG medium and cultured overnight at 28°C in a shaker. Centrifuge at 4°C, 5000g for 5 minutes, discard the excess LB, and resuspend the bacteria in tobacco instant resuspension solution to make the bacterial liquid OD 600 The results showed that compared with NIB1, NIB1 ΔIDR With NIB1 R49A With NIB1 G50A The ability to accelerate necrosis of NVR1 was weakened ( Fig. 3 , D, H, K, and H).

[0155] Example 4: NIB1 can recruit EDS1 to aggregates

[0156] a: BiFC validation of the interaction between NIB1 and EDS1

[0157] The proteins EDS1 and NIB1, used to verify their interaction, were linked to the MSH21-35S-cGFP and MSH22-35S-nGFP vectors, respectively. After sequencing, the two vectors were transformed into Agrobacterium tumefaciens GV3101. After two days of culture on solid RKG medium at 28°C, single colonies were isolated and plated in 4 mL of RKG liquid medium. After overnight culture at 28°C, tobacco leaves were transformed and labeled according to the transient transfection protocol. Two days later, the leaves were observed under a fluorescence confocal microscope and images were acquired, demonstrating an interaction between NIB1 and EDS1 (Figure 4, D).

[0158] b: Co-localization observation of NIB1 recruiting EDS1 to nuclear condensates

[0159] NIB1 and EDS1 were linked to vectors carrying YFP and mCherry, respectively. After sequencing, each vector was transformed into Agrobacterium tumefaciens GV3101. After two days of culture on RKG solid medium at 28°C, a single colony was picked and transferred to 4 mL of RKG liquid medium. After overnight culture at 28°C, the colony was transformed into tobacco leaves at a 1:1 ratio and labeled. Microscopic observation two days later revealed that NIB1-YFP recruited EDS1-mCherry to bright bodies in the nucleus (Figure 4, E).

[0160] Example 5: NIB1 positively regulates plant disease and insect resistance

[0161] a: Rice blast fungus in vitro experiment

[0162] The preserved TH12 rice blast fungus (laboratory preservation) was inoculated into CM solid medium and cultured at 28°C for 10 days. The spores were washed with sterile water and then counted using a hemocytometer. The spore concentration was adjusted to 6 × 10 5 spores / mL. Rice leaves at the tillering stage were cut into approximately 10 cm long segments. The leaves were then placed in agar-solidified culture dishes, with both ends carefully moisturized to prevent water loss. The leaf surface was scratched with a 1 mL syringe needle, followed by the addition of 10 μL of spore solution. The leaves were then placed in an inoculation box. As shown in Figures 5, AC, NIB1 positively regulates plant resistance to rice blast.

[0163] CM medium recipe: 20× Nitrate salts (50 mL), 1000× Trace elements (1 mL), 1000× Vitamin solution (1 mL), D-glucose 10 g, Peptone 2 g, Yease extract 1 g, Casamino acid 1 g. Adjust pH to 6.5 with 10 M NaOH, add ddH₂O to 1000 mL, and autoclave at 115°C for 20 min. For solid medium, add 15 g agar powder per liter.

[0164] The NIB1-OE transgenic plants were provided by Wuhan Boyuan Biological, which was also responsible for the experimental processes including vectors, Agrobacterium, rice lines, transformation, cultivation, and subculture.

[0165] b: Experiment on rice leaf roller

[0166] Rice leaves at the tillering stage were cut into approximately 10 cm long segments and placed in Petri dishes lined with moistened gauze, with approximately seven leaves per dish. Third-instar nymphs of the rice leaf roller (collected from Songjiang Farm in Shanghai) were then placed on the rice leaves, with paper blocks placed on either side. Water was added daily to prevent the rice from drying out. Three days later, the insects were weighed. As shown in Figure 5 (DF), insects feeding on NIB1-KO plants weighed significantly more compared to the control and NIB1-OE (NIB1 overexpressing) plants.

[0167] Table 1: Primers used in this study

[0168] sequence of this article

[0169] SEQ ID NO: 1—NIB1 gene nucleotide sequence

[0170] SEQ ID NO: 2—NIB1 gene amino acid sequence

Claims

1. Use of the NIB1 gene or its encoded polypeptide or promoter in enhancing NVR1 activity in plants, recruiting EDS1 to the nucleus, or upregulating plant disease and insect resistance.

2. The use according to claim 1, characterized in that The disease and insect resistance is selected from one or more of the following: resistance to rice leaf roller, resistance to cotton bollworm, resistance to rice blast fungus, and / or The promoter is selected from the group consisting of small molecule compounds, nucleic acid molecules, or a combination thereof.

3. The use according to claim 1 or 2, characterized in that The plant is a grass plant, Preferably, the grass plant is rice, barley, wheat, oats or rye.

4. The use according to claim 1 or 2, characterized in that The polypeptide encoded by the NIB1 gene is selected from the following group: (a) a polypeptide having the sequence shown in SEQ ID NO: 2, (b) a polypeptide derived from (a) formed by substituting, deleting or adding one or more amino acid residues in the sequence shown in SEQ ID NO: 2 and having the function of the polypeptide of (a), or (c) A polypeptide derived from (a) that has a sequence homology of more than 90% with the polypeptide of (a) and has the function of the polypeptide of (a).

5. The use according to claim 1 or 2, characterized in that The nucleic acid sequence of the NIB1 gene is selected from: (1) a polynucleotide sequence as shown in SEQ ID NO: 1 or a polynucleotide sequence having 80% or more homology thereto, (2) a polynucleotide sequence having 1 to 60 nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide sequence shown in SEQ ID NO: 1, (3) A polynucleotide sequence complementary to the polynucleotide sequence described in any one of (1) to (2).

6. A method for inactivating a NIB1 protein, comprising mutating the amino acid at position 49 and / or 50, wherein the mutation is preferably a substitution or deletion mutation; more preferably, the R at position 49 is mutated to an A, and / or the G at position 50 is mutated to an A.

7. A method for enhancing the activity of NVR1 in plants, recruiting EDS1 to the cell nucleus, and regulating the plant's ability to resist pests and diseases, the method comprising the steps of: upregulating the expression or activity of the NIB1 gene in the plant, Preferably, the disease and insect resistance is selected from one or more of the following: resistance to rice leaf roller, resistance to cotton bollworm, and resistance to rice blast fungus.

8. The method according to claim 7, wherein The plant is a grass plant, Preferably, the grass plant is rice, barley, wheat, oats, rye, More preferably, the grass plant is rice.

9. The method according to claim 7 or 8, wherein The step of upregulating the expression or activity of the NIB1 gene in the plant comprises: transferring the NIB1 gene into the plant to obtain a transformed plant, Preferably, the step of upregulating the expression or activity of the NIB1 gene in the plant comprises: contacting the plant cells, tissues or organs with Agrobacterium containing the nucleic acid construct of the NIB1 gene, thereby transferring the nucleic acid construct into the plant cells, tissues or organs; More preferably, the nucleic acid construct is an expression vector or a recombinant vector. More preferably, the method for upregulating the expression of the NIB1 gene in a plant further comprises: selecting plant cells, tissues, organs or seeds into which the NIB1 gene has been introduced, and regenerating the plant cells, tissues, organs or seeds into a plant.

10. Use of the NIB1 gene as a target in screening for potential agents that can increase NVR1 activity in plants, recruit EDS1 to the nucleus, and enhance plant resistance to pests and diseases.

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