CN120519468A

CN120519468A - Application of NIB1 gene in plant disease and insect resistance

Info

Publication number: CN120519468A
Application number: CN202410200778.0A
Authority: CN
Inventors: 陈晓亚; 郭晓祥; 黄嘉发; 赵靖童; 黄金泉; 王凌健; 陈梅
Original assignee: Center for Excellence in Molecular Plant Sciences of CAS
Current assignee: Center for Excellence in Molecular Plant Sciences of CAS
Priority date: 2024-02-22
Filing date: 2024-02-22
Publication date: 2025-08-22
Also published as: WO2025176010A1

Abstract

The invention relates to application of an NIB1 gene in plant disease and insect resistance, and particularly provides application of the NIB1 gene or a coded polypeptide or an accelerant thereof in enhancing activity of NVR1 in plants, recruiting EDS1 to cell nuclei or up-regulating plant disease and insect resistance. The invention enhances the plant resistance to diseases and insects by up-regulating the expression level of NIB 1.

Description

Application of NIB1 gene in plant disease and insect resistance

Technical Field

The invention relates to the fields of biotechnology and botanic, in particular to a function of rice RNA binding protein in resisting diseases and insects and application thereof.

Background

In organisms, RNA Binding Proteins (RBPs) act as important post-transcriptional regulatory factors, regulating RNA metabolism by binding to RNA. The N-terminal of traditional glycine-rich RNA binding proteins (GR-RBPs) contains an RNA recognition motif (RNA recognition motif, RRM) or a cold shock domain (cold-shock domain), and the C-terminal is a glycine-rich domain (glycine-rich domain), so that the protein participates in RNA splicing, transportation and editing, plays a very important role in plant response to cold stimulus, ultraviolet radiation, saline-alkali, pathogen infection and the like (Ma et al, 2021a; sachetto-Martins et al, 2000). This class of proteins constitutes the GRPIV family of GRP superfamilies (Ciuzan et al, 2015). In plant-pathogen interactions, the molecular mechanisms by which GR-RBPs recognize pathogen effectors and the interaction of GR-RBPs and NLR receptors have been studied intensively. Pseudomonas syringae type III effector HopU1 can target Arabidopsis AtGRP7, weaken RNA binding capacity of the Arabidopsis through ribosylation modification of arginine 49 th position of RRM in AtGRP, weaken promotion effect of the Arabidopsis on downstream defense reaction, and inhibit immune response of plants (Fu et al, 2007; jeong et al, 2011). CaGRP1 of Capsicum annuum (Capsicum annuum) regulates CaPIK1 expression negatively through RRM domain interaction with CaPIK1 (receiver-like cytoplasmic protein kinase 1), inhibits CaPIK-induced ROS and cell death, whereas CaGRP 1-silenced plants up-regulate CaPIK1, caPR1 and CaDEF1 expression, thereby increasing resistance of Capsicum to Xanthomonas campestris (Xanthomonas campestris pv.Vesicaria, xcv) (Kim et al, 2015). The interaction of NbGRP and CNL receptor Gpa2 in Nicotiana benthamiana improves the resistance to Solanum tuberosum (Globodera pallida), and the RRM domain can maintain Rx1 (PVX immunoreceptor) steady state, and has a certain relationship with plant immunity related to PVX infection (Sukarta, etc., 2022). Functional studies on how plant RNA binding proteins recognize insect secreted nucleases and their catalytic products in disease and insect resistance have not been reported.

Disclosure of Invention

The inventors provide for the first time a method for regulating the ability of plants to resist disease and insects by regulating the expression level of the NIB1 gene in plants of the poaceae family to regulate the plant's defensive response.

In a first aspect, the invention provides the use of the NIB1 gene or a polypeptide or promoter encoding the same to enhance the activity of NVR1 in plants, recruit EDS1 to the nucleus or up-regulate plant resistance to disease and insects.

In one or more embodiments, the disease and pest resistance is selected from one or more of rice leaf rollers, cotton bollworms, rice blast bacteria.

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.

In one or more embodiments, the plant is a gramineous plant.

In one or more embodiments, the gramineous plant is rice, barley, wheat, oat, rye. Preferably, the graminaceous plant is rice.

In one or more embodiments, the NIB1 gene is from a graminaceous plant, preferably from rice.

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

(a) A polypeptide having a sequence shown in SEQ ID NO. 2;

(b) A polypeptide derived from (a) and having the function of (a) and formed by substitution, deletion or addition of one or more (e.g., 1 to 20; preferably 1 to 10; more preferably 1 to 5) amino acid residues to the sequence shown in SEQ ID NO. 2, or

(C) A polypeptide derived from (a) having more than 90% (preferably 93%; more preferably 95% or 98%) homology to the polypeptide sequence of (a) and having the function of the polypeptide of (a).

In one or more embodiments, the nucleic acid sequence of the NIB1 gene is selected from the group consisting of:

(1) The polynucleotide sequence shown in SEQ ID NO. 1 or a polynucleotide sequence having 80% (preferably 90%; more preferably 95% or 98%) or more homology thereto;

(2) A polynucleotide sequence of 1 to 60, preferably 1 to 30, more preferably 1 to 10 nucleotides truncated or added at the 5 'and/or 3' end of the polynucleotide sequence shown in SEQ ID NO. 1;

(3) A polynucleotide sequence complementary to the polynucleotide sequence of any one of (1) to (2).

The invention also provides a method of inactivating an NIB1 protein comprising mutating amino acids 49 and/or 50 thereof. The NIB1 protein has a sequence shown in SEQ ID NO. 2.

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

In one or more embodiments, R at position 49 is mutated to a.

In one or more embodiments, the G at position 50 is mutated to a.

In a second aspect, the invention provides a method of enhancing activity of NVR1 in a plant, recruiting EDS1 to the nucleus, and enhancing disease and pest resistance in a plant, comprising the step of up-regulating expression or activity of the NIB1 gene in the plant. Preferably, the disease and pest resistance is selected from one or more of rice leaf rollers, cotton bollworms and rice blast bacteria.

In one or more embodiments, the plant is a gramineous plant.

In one or more embodiments, the gramineous plant is rice, barley, wheat, oat, rye. Preferably, the gramineous plant is rice.

(a) A polypeptide having a sequence shown in SEQ ID NO. 2;

In one or more embodiments, the step of up-regulating the expression or activity of the NIB1 gene in the plant comprises transferring the NIB1 gene into the plant to obtain a transformed plant.

In one or more embodiments, the step of up-regulating the expression or activity of the NIB1 gene in a plant comprises:

(1) Contacting a cell or tissue or organ of a plant with agrobacterium containing a nucleic acid construct of the NIB1 gene, thereby transferring the nucleic acid construct into the plant cell, tissue or organ.

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

In one or more embodiments, the method of up-regulating expression of the NIB1 gene in a plant further comprises:

(2) Selecting a plant cell, tissue, organ or seed into which the NIB1 gene has been transferred, and

(3) Regenerating the plant cells, tissues, organs or seeds of step (3).

The invention also provides application of the NIB1 gene as a target in screening potential agents, wherein the potential agents can improve activity of NVR1 in plants, recruit EDS1 to cell nuclei and enhance plant insect resistance.

In one or more embodiments, the screening includes the steps of (1) contacting a candidate agent with a NIB1 protein or a nucleic acid molecule encoding the same or a system containing the same, and (2) detecting a change in expression or activity of the protein or nucleic acid molecule encoding the same, which may be indicative of the candidate agent as a potential agent of interest. If the candidate agent increases the expression or activity of the NIB1 protein or nucleic acid molecule encoding the same, it is indicated that the candidate agent is a potential agent that increases the activity of NVR1 in plants, recruits EDS1 to the nucleus, and enhances plant disease and pest resistance. In a preferred embodiment, step (1) comprises adding a candidate agent to a system comprising NIB1 protein or a nucleic acid molecule encoding the same in a test set, and step (2) comprises detecting the expression or activity of NIB1 protein in the system of the test set, and comparing it to a control set, wherein the control set 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, e.g., 20% higher, preferably 50% higher, more preferably 80% higher) than that in the control group, this candidate is indicative of a potential agent that increases NVR1 activity in plants, recruits EDS1 to the nucleus, and enhances plant resistance to disease and pest.

In one or more embodiments, the screening comprises the steps of (1) contacting a candidate agent, a NIB1 protein or nucleic acid molecule encoding thereof, with (a) a NVR1 protein or nucleic acid molecule encoding thereof and/or (B) an EDS1 protein or nucleic acid molecule encoding thereof, or (C) a system containing (a) and/or (B), and (2) indicating that the candidate agent is a potential agent that increases the activity of NVR1 in plants, recruits EDS1 to the nucleus, increases plant disease and pest resistance, if any one or more of (A) increases the interaction of the NIB1 protein with the NVR1 protein, (B) increases the activity of the NVR1 protein, (C) increases the interaction of the NIB1 protein with the EDS1 protein, and (D) increases the recruitment of EDS1 to the aggregate by NIB 1.

In one or more embodiments, the screening comprises the steps of (1) adding a candidate agent to a system comprising a NIB1 protein or nucleic acid molecule encoding thereof and a NVR1 protein or nucleic acid molecule encoding thereof in a test set, (2) detecting the activity of the NVR1 protein or the interaction of the NIB1 protein with the NVR1 protein in the system of the test set, and comparing to a control set, wherein the control set is the same system without the addition of the candidate agent. If the activity of the NVR1 protein or the interaction of the NIB1 protein with the NVR1 protein in the test group is statistically higher (preferably significantly higher, such as 20% higher, preferably 50% higher, more preferably 80% higher) than that in the control group, this candidate is shown to be a potential agent that increases the activity of NVR1 in plants, recruits EDS1 to the nucleus, and enhances plant disease and pest resistance.

In one or more embodiments, the screening includes the steps of (1) adding a candidate agent to a system comprising a NIB1 protein or nucleic acid molecule encoding thereof and an EDS1 protein or nucleic acid molecule encoding thereof in a test set, (2) detecting interaction of the NIB1 protein with the EDS1 protein or EDS1 protein in an aggregate in the system of the test set, and comparing to a control set, wherein the control set is the same system without the addition of the candidate agent. If the interaction of NIB1 protein with EDS1 protein or the EDS1 protein content in the aggregate in the test group is statistically higher (preferably significantly higher, such as 20% higher, preferably 50% higher, more preferably 80% higher) than that in the control group, this candidate is shown to be a potential agent that increases NVR1 activity in plants, recruits EDS1 to the nucleus, and enhances plant disease and pest resistance.

In one or more embodiments, the system is selected from a cell system (e.g., 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.

In one or more embodiments, the interaction between proteins is detected by a MST assay, co-IP, or a bimolecular luciferase complementation assay.

In one or more embodiments, protein expression is detected by detecting an antibody antigen reaction, detecting mRNA content, e.g., western, ELISA or Southern.

In one or more embodiments, EDS1 protein content in the aggregate is detected by microscopic co-localization observation. For example, NIB1 and EDS1 were separately ligated onto carriers with different fluorescent markers, and the tobacco leaves were transformed and then observed for microscopic co-localization.

Drawings

Fig. 1, NIB1 interacts with NVR 1.

A, MST experiments showed that NIB1 binds directly to NVR1, where NIB1 is a ligand.

B, in-half Pull down experiments demonstrated that NIB1 interacted with NVR 1.

C, co-IP verifies that NVR1 interacts with NIB 1.

D, NIB1 protein structure schematic diagram. RNA Recognition Motif, RNA recognition motif, glycine-rich region, glycine-rich domain.

E, bimolecular luciferase complementation experiments verify that the interaction of NIB1 and NVR1 is dependent on the RRM domain in tobacco.

FIG. 2 amino acid residues 49 and 50 of NIB1 are important for NIB 1.

A, NIB1 results are shown schematically with arginine at position 49 and glycine at position 50.

B, molecular docking results show that NIB1 can bind to 2',3' -cAMP, and the amino acids responsible for recognition are R49 and G50.

C, the amino acid that binds 2',3' -cGMP is G50.

D, MST experiments demonstrated that NIB1 binds 2',3' -cAMP, and that NIB1 ^R49A binds less and loses its ability to bind NIB1 ^G50A.

E, fluorescence confocal microscopy showed that aggregates could form in the nuclei of NIB1-YFP cells in the presence of NVR1, and that NIB1 ^R49 and NIB1 ^G50A could not.

F, fluorescence confocal microscopy showed the ability of 2',3' -cAMP to promote the formation of aggregates in the nuclei of NIB1-YFP, which is lost by NIB1 ^R49 and NIB1 ^G50A. The scale bars are all 5 microns.

G, transient expression of NIB1-YFP in tobacco leaves can cause plant cell death. NIB1 ^R49A and NIB1 ^G50A do not cause necrosis of plant cells.

H, cotton bollworm feeding experiments show that transient expression of NIB1 positively regulates cotton bollworm resistance.

FIG. 3 NIB1 accelerates NVR1 activity.

A, urea SDS-PAGE detects the effect of NIB1-YFP and NIB1 ^ΔIDR on NVR1 catalytic activity.

B-C, HPLC-MS characterizes the effect of NIB1-YFP and NIB1 ^ΔIDR on NVR1 catalytic activity by detecting 2',3' -cAMP/cGMP.

And D, expressing NVR1-HA+YFP, NVR1-HA+NIB1-YFP and NVR1-HA+NIB1 ^ΔIDR -YFP respectively in tobacco leaves by using an agrobacterium-mediated tobacco transient expression system, observing necrosis after 3 days, and taking a picture. The scale is 1 cm.

E-H quantifies A-D, respectively.

I-J, HPLC-MS characterizes NIB1-YFP and the effect of NIB1 ^R49A and NIB1 ^G50A on NVR1 catalytic activity by detecting 2',3' -cAMP/cGMP.

K-L, NVR1+NIB1 ^R49A and NVR1+NIB1 ^G50A were expressed by an Agrobacterium-mediated tobacco transient transformation system and three days later, sampled and photographed.

FIG. 4 NIB1 is capable of recruiting EDS1 to the aggregate.

A, the bimolecular luciferase complementation experiment verifies that NIB1 interacts with EDS 1. The red-blue gradient indicates the interaction strength.

B, semi-in vitro Pull-Down experiments verify that His-NIB1 interacts with EDS 1-Flag. Immunoblot hybridization was performed using His and Flag antibodies, respectively.

C, in tobacco leaves, NIB1-Flag was verified to interact with EDS1-GFP using co-immunoprecipitation. Hybridization was performed using Flag and GFP antibodies, respectively.

And D, in the tobacco leaves, using a bimolecular fluorescence complementation experiment BiFC to verify the interaction between EDS1 and NIB 1. The scale is 20 microns.

E, co-localization results showed that NIB1 recruited EDS1 into the aggregate. The scale is 5 microns.

FIG. 5 NIB1 positively regulates the disease and pest resistance of plants.

A, the onset of ZH11, NIB1-KO and NIB1-OE plants 5 days after inoculation with Pyricularia oryzae (TH 12).

B, the length of the lesion after 5 days of infection of the rice blast bacteria. A total of 11 leaves were counted.

C, qRT-PCR calculates the relative quantity of rice blast germ by pathogenic bacterium 28S rDNA through rice reference gene ACTIN.

D, insect resistance of ZH11, NIB1-KO and NIB1-OE plants after 5 days of inoculation of cnaphalocrocis medinalis.

E, feeding rice leaf rollers after three days.

And F, weighing the cnaphalocrocis medinalis.

Detailed Description

The inventor firstly reveals that the capability of obviously regulating the plant defense response by directionally regulating and controlling the expression level of the NIB1 gene in the gramineous plant, thereby regulating and controlling the disease and insect resistance of the plant, including the capability of resisting cnaphalocrocis medinalis, cotton bollworm and rice blast.

As used herein, a "graminaceous plant" is rice, barley, wheat, oat, rye.

In the present invention, the term "NIB1" refers to a polypeptide having the sequence of SEQ ID NO:2 having NIB1 activity or a polypeptide encoded by the NIB1 gene having the ID LOC_Os03g 46770. The term also includes variants of the SEQ ID NO:2 sequence having the same function as NIB 1. Such variants include, but are not limited to, deletions, insertions and/or substitutions of several (typically 1-50, preferably 1-30, 1-20, 1-10, 1-8, 1-5) amino acids, and additions or deletions of one or several (typically within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. Amino acids of similar properties are often referred to in the art as 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), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, lactic acid, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). As another example, the addition of one or more amino acids at the amino-and/or carboxy-terminus typically does not alter the function of the polypeptide or protein. Conservative amino acid substitutions for many commonly known non-genetically encoded amino acids are known in the art. Conservative substitutions of other non-coding amino acids may be determined based on a comparison of their physical properties with those of the genetically encoded amino acid.

Variant forms of the polypeptide include homologous sequences, conservative variants, allelic variants, natural mutants, and induced mutants.

Any polypeptide having a high homology to the NIB1 (e.g., a homology of 70% or more to the sequence shown in SEQ ID NO: 2; preferably 80% or more; more preferably 90% or more, e.g., a homology of 95%,98% or 99%) and having a similar or identical function to NIB1 is also included in the present invention. The term "same or similar function" refers mainly to the regulation of the disease and pest resistance of crops (e.g. rice).

The invention also includes analogs of the claimed polypeptides. These analogs may differ from the native SEQ ID NO. 2 by differences in amino acid sequence, by differences in modified forms that do not affect the sequence, or by both. Analogs of these proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis or other known biological techniques. Analogs also include analogs having residues other than the natural L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It should be understood that the proteins of the present invention are not limited to the representative proteins exemplified above.

Modified (typically without altering the primary structure) forms include chemically derivatized forms of the protein in vivo or in vitro such as acetylation or carboxylation. Modifications also include glycosylation, such as those that are glycosylation modified during protein synthesis and processing. Such modification may be accomplished by exposing the protein to an enzyme that performs glycosylation (e.g., mammalian glycosylase or deglycosylase). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine).

The polypeptide fragments, derivatives or analogues of the invention may be (i) a polypeptide having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein. Such fragments, derivatives and analogs are within the purview of one skilled in the art in view of the definitions herein.

In addition, any biologically active fragment of NIB1 may be used in the present invention. By biologically active fragment of NIB1 is meant herein as a polypeptide which is still capable of retaining all or part of the functionality of full length NIB 1. Typically, the biologically active fragment retains at least 50% of the activity of full length NIB 1. Under more preferred conditions, the active fragment is capable of retaining 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of full-length NIB 1.

The invention also relates to polynucleotide sequences encoding the NIB1 of the invention or variants, analogs, 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.

The invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention. Variants of the polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. Such nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded polypeptide. As used herein, degenerate variants refer to nucleic acid sequences which encode a protein having SEQ ID NO.2, but which differ from the coding region sequence shown in SEQ ID NO. 1. A "polynucleotide encoding a polypeptide" may be a polynucleotide that includes a polynucleotide encoding the polypeptide, or may also include additional coding and/or non-coding sequences.

The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. As used herein, "stringent conditions" means (1) hybridization and elution at a relatively low ionic strength and a relatively high temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃, or (2) hybridization with a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42 ℃, etc., or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably 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.

It will be appreciated that although the genes provided in the examples of the present application are derived from rice, the gene sequences of NIB1 which are derived from other similar plants (especially plants belonging to the same family or genus as rice) and which have a certain homology (e.g.more than 70%, such as 80%,85%, 90%, 95%, or even 98% sequence identity) to the sequences of the present application (preferably as shown in SEQ ID NO: 1) are included within the scope of the present application, as long as the person skilled in the art can readily isolate the sequences from other plants based on the information provided in accordance with the present application after reading the present application. Methods and tools for aligning sequence identity are also well known in the art, such as BLAST.

The full length sequence of the NIB1 nucleotide or a fragment thereof of the present invention can be generally obtained by PCR amplification, recombinant or artificial synthesis. For the PCR amplification method, primers can be designed according to the nucleotide sequences disclosed in the present invention, particularly the open reading frame sequences, and amplified to obtain the relevant sequences using a commercially available DNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order. Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. It is usually cloned into a vector, transferred into a cell, and then isolated from the proliferated host cell by a conventional method to obtain the relevant sequence.

Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.

The invention also provides a nucleic acid construct comprising the gene of the invention. As a preferred mode, the promoter downstream of the nucleic acid construct comprises a multiple cloning site or at least one cleavage site. When it is desired to express the gene of interest of the present invention, the gene of interest is ligated into a suitable multiple cloning site or cleavage site, thereby operably linking the gene of interest to a promoter. As another preferred mode, the nucleic acid construct comprises (in the 5 'to 3' direction) a promoter, a gene of interest, and a terminator. The nucleic acid construct may further comprise, if desired, elements selected from the group consisting of 3' polynucleotide signals, untranslated nucleic acid sequences, transport and targeting nucleic acid sequences, resistance selection markers (dihydrofolate reductase, neomycin resistance, hygromycin resistance, and green fluorescent protein, etc.), enhancers, or operators.

Methods for preparing nucleic acid constructs are well known to those of ordinary skill in the art. The expression vector may be a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. In general, any plasmid or vector may be used as long as it is capable of replication and stability in a host.

One of ordinary skill 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 recombinant techniques, and the like. When the gene of the present invention is used to construct recombinant expression vectors, any one of enhanced, constitutive, tissue-specific or inducible promoters may be added before the transcription initiation nucleotide.

The inclusion of the gene or nucleic acid construct of the invention may be used to transform an appropriate host cell to allow the host to express the protein. The host cell may be a prokaryotic cell such as E.coli, streptomyces, agrobacterium, or a lower eukaryotic cell such as a yeast cell, or a higher eukaryotic cell such as a plant cell. It will be clear to one of ordinary skill in the art how to select appropriate vectors and host cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote (e.g., E.coli), the treatment may be performed by CaCl ₂ or electroporation. When the host is eukaryotic, DNA transfection methods such as calcium phosphate co-precipitation, conventional mechanical methods (e.g., microinjection, electroporation, liposome packaging, etc.) may be used. The transformed plant may also be transformed by Agrobacterium or gene gun, such as leaf disc method, embryo transformation method, flower bud soaking method, etc. Plants can be regenerated from the transformed plant cells, tissues or organs by conventional methods to obtain transgenic plants. When expressed in higher eukaryotic cells, the polynucleotide will have enhanced transcription if inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase the transcription of a gene.

It will be clear to a person of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

The polypeptides described herein may be expressed within a cell, or on a cell membrane, or secreted outside of a cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.

Transformation of the host with the recombinant DNA may be carried out by conventional techniques well known to those skilled in the art. The transformed plants may be transformed by Agrobacterium or gene gun, for example, spraying, leaf disc, young embryo transformation, etc. Plants can be regenerated from the transformed plant tissue or organ by conventional methods to obtain plants with altered traits.

The invention provides application of the NIB1 gene, which is used for regulating and controlling the activity of NVR1 in plants, recruiting EDS1 to cell nuclei or regulating and controlling the disease and insect resistance of the plants. As a preferred mode, the NIB1 gene can be used for enhancing the activity of NVR1 in plants and increasing the disease and pest resistance of the plants.

The invention also relates to an NIB1 promoter and application thereof, and the promoter of the NIB1 can improve the expression and/or activity of the NIB1, so that the promoter can regulate and control the activity of NVR1 in plants and the defense reaction of the plants through the influence on the NIB1, thereby achieving the plant disease and insect resistance.

In one aspect, any agent that increases the activity, increases the stability, promotes the expression, increases the effective duration, or promotes the transcription and translation of the NIB1 gene may be used in the present invention as an "enhancer" of the NIB1 gene for regulating plant disease and pest resistance. Such as expression vectors that increase transcription, expression or activity of the NIB1 gene.

The invention also provides a method for regulating and controlling the activity of NVR1 in plants, recruiting EDS1 to cell nuclei or regulating and controlling the disease and pest resistance of plants, which comprises the steps of up-regulating the expression or activity of NIB1 genes in plants, thereby regulating and controlling the activity of NVR1 in plants and regulating and controlling the disease and pest resistance of plants. After the use of the NIB1 gene is known, various methods well known to those skilled in the art may be employed to up-regulate the expression of the NIB1 gene. For example, expression units carrying the NIB1 gene (e.g., expression vectors or viruses, etc.) can be delivered to the target site and allowed to express active NIB1 by means known to those skilled in the art.

As one embodiment of the present invention, the NIB1 gene is cloned into an appropriate vector by a conventional method, and the nucleic acid construct with the NIB1 gene is introduced into a plant tissue or organ to cause the plant to express the NIB1 gene. Plants that overexpress the NIB1 gene may be obtained by regenerating the plant tissue or organ into a plant.

In one or more embodiments, the method of up-regulating expression of the NIB1 gene comprises:

(1) Providing an Agrobacterium harboring a nucleic acid construct comprising the NIB1 gene,

(2) Contacting a cell or tissue or organ of a plant with the agrobacterium of step (1), thereby transferring the nucleic acid construct into the plant tissue or organ.

(3) Selecting a plant tissue, organ or seed into which the NIB1 gene has been transferred, and

(4) Regenerating the plant tissue, organ or seed of step (3).

In this context, the screening comprises the steps of (1) contacting a candidate agent with a NIB1 protein or a nucleic acid molecule encoding the same or a system containing the same, and (2) detecting a change in the expression or activity of the protein or nucleic acid molecule encoding the same, which may be indicative of the candidate agent as the potential agent of interest. If the candidate agent increases the expression or activity of the NIB1 protein or nucleic acid molecule encoding the same, it is indicated that the candidate agent is a potential agent that increases the activity of NVR1 in plants, recruits EDS1 to the nucleus, and enhances plant disease and pest resistance. In a preferred embodiment, step (1) comprises adding a candidate agent to a system comprising NIB1 protein or a nucleic acid molecule encoding the same in a test set, and step (2) comprises detecting the expression or activity of NIB1 protein in the system of the test set, and comparing it to a control set, wherein the control set 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, e.g., 20% higher, preferably 50% higher, more preferably 80% higher) than that in the control group, this candidate is indicative of a potential agent that increases NVR1 activity in plants, recruits EDS1 to the nucleus, and enhances plant resistance to disease and pest.

As used herein, the "system" is selected from a cell system (e.g., 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. In one or more embodiments, the interaction between proteins is detected by a MST assay, co-IP, or a bimolecular luciferase complementation assay. In one or more embodiments, protein expression is detected by detecting an antibody antigen reaction, detecting mRNA content, e.g., western, ELISA or Southern. In one or more embodiments, EDS1 protein content in the aggregate is detected by microscopic co-localization observation. For example, NIB1 and EDS1 were separately ligated onto carriers with different fluorescent markers, and the tobacco leaves were transformed and then observed for microscopic co-localization.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein. The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by conventional conditions such as those described in Sambrook et al, molecular cloning, A laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or by the manufacturer's recommendations.

Examples

Example 1 NIB1 interacts with NVR1

MST detection of NIB1 interaction with NVR1

And (3) carrying out RED-NHS labeling on the purified NIB1 protein by using a kit (RED-NHS protein labeling kit (amino label-RED CHANNEL) and NT-L111) to obtain NHS-NIB1 protein, and detecting whether the protein is aggregated or not and whether capillary adsorption phenomenon exists or not by detecting the NHS-NIB1 protein, and if no adsorption or aggregation exists, carrying out subsequent experiments. Tween may be added in proper amount for adsorption and aggregation, but the concentration is not too high. Ligand (E.coli purified NVR1 protein) was diluted by first preparing 20. Mu.L of protein (20-50 times the Kd concentration), adding 10. Mu.L of PBS to 15 PCR tubes, sequentially diluting the protein concentration, adding NHS-NIB1 protein to the above PCR tubes, and blowing uniformly, and detecting by adsorbing the sample with a capillary (FIG. 1, A).

B in-half Pull-Down experiments

NIB1 was constructed on pEAQ-flag vector (stored in this laboratory), and after successful sequencing, agrobacterium was transformed by thawing Agrobacterium competent GV3101 (organism only) on ice to ice water mix, and adding 1. Mu.L of plasmid with fragment of interest to competent cells. Placing on ice for 30min, cooling in liquid nitrogen for 30s, placing in a 37 ℃ water bath for 5min, ice-bathing for 3min, adding 100 mu L of liquid LB culture medium into competent cells, and shake culturing in a 28 ℃ incubator for 3h. And (3) 50 mu L of bacterial liquid is taken and coated on a corresponding resistant solid culture medium, the culture is carried out for 48 hours at the temperature of 28 ℃, and the monoclonal is selected to obtain the agrobacterium which is successfully transformed.

The method comprises the steps of instantaneously transforming tobacco, namely picking up a monoclonal in liquid RKG culture medium one night in advance, centrifuging bacterial liquid cultured overnight, 5000rpm for 5min, carefully pouring out supernatant culture medium, adding 1mL of heavy suspension into the precipitate, and blowing and beating the heavy suspension by a gun head to obtain uniformly heavy suspension bacterial liquid. The nanometer Drop is used for measuring OD ₆₀₀ nm, the concentration of bacterial liquid is regulated by using heavy suspension, the OD ₆₀₀ nm of single bacterial liquid is about 0.6, the OD ₆₀₀ nm of mixed injection of two bacterial liquids is about 1, the OD ₆₀₀ nm of single bacterial liquid is ensured to be about 0.5 after mixing, and the tobacco leaves are injected by using a syringe after removing a needle head and marked.

Extracting total protein of tobacco leaves after two days, namely placing 0.25g of tobacco leaves into a 2mL centrifuge tube containing steel balls, quick-freezing the tobacco leaves in liquid nitrogen, completely crushing the leaves in a crusher, adding 1mL of IP-lysis buffer into the tube, vortex oscillating for 1min, ice bath for 5min, repeating for 3-5 times, centrifuging for 10min at 14000rpm, taking supernatant into a new centrifuge tube, centrifuging for 10min at 14000rpm, repeating for two to three times, obtaining the supernatant which is the total protein liquid of the tobacco, and carrying out different experiments after measuring the concentration. At the same time, purification of another His-tagged protein NVR1 is required.

Mu.L of the nickel column to which His recombinant protein had been bound was taken into a 1.5mL centrifuge tube while 800. Mu.L of the extracted total protein of tobacco was added and incubated at 4℃for 3h with rotation. The protein solution after the incubation was centrifuged at 2500rpm at 4℃for 3min and the supernatant carefully aspirated. mu.L of Co-IP buffer (50 mM Tris-HCl,150mM NaCl,20% glycerol, 0.5% NP-40,1 XPIC (full gold)) was added to the centrifuge tube, spun for 5min, centrifuged at 2500rpm at 4℃for 3min, the supernatant carefully aspirated, repeated 5 times, 40. Mu.L of 4 XProtein Loading buffer (Tanon, 180-8210D) was added, and 20. Mu.L was taken and then subjected to Western immunoblotting using Flag antibody (ZSGB-BIO, TA-05). Meanwhile, his recombinant protein and tobacco leaf protein combined on a nickel column are taken as Input, and an immunoblotting experiment is carried out by utilizing His and Flag antibodies respectively (figure 1, B).

Co-IP verifies in vivo interaction of NVR1 with NIB1

The method comprises the steps of respectively connecting proteins to be verified for interaction, NVR1 and NIB1 with pCAMBIA1300-YFP (preserved in a laboratory) and pEAQ-FLAG vectors, respectively transforming agrobacterium GV3101, and performing transient transformation steps of mixed tobacco of two bacterial liquids. After 2 days, total tobacco leaf protein was extracted, 30. Mu.L GFP beads (Chromotek, gta-20) was added to the total protein and incubated overnight at 4℃with rotation. The next day was centrifuged at 2500rpm for 3min, the supernatant was gently aspirated, 500. Mu. L IP Lysis buffer (50 mM Tris-HCl,150mM NaCl,20% glycerol, 0.5% NP-40,1 XPIC (full gold)) was added, the incubation was rotated for 5min, and the centrifugation at 2500rpm was repeated 5 times for 3min, whereby protein interaction was detected by Western immunoblotting using Flag antibody. The total protein was also detected as input using Flag and GFP antibodies, respectively (fig. 1, c).

D, double-molecule luciferase complementary experiment NIB1 combined with NVR1

Proteins NVR1 and NIB1 for verifying interaction were ligated to the JW772-35S-cLUC vector (stored in this laboratory) and the JW771-35S-nLUC vector (stored in this laboratory), respectively. After sequencing was correct, both vectors were transformed into Agrobacterium GV3101, respectively, and after culturing for two days at 28℃on a solid medium containing RKG (tryptone 10g, yeast extract 5g, naCl10g, agar 15g, tryptone 10g, yeast extract 5g, naCl10g, agar 15g (solid), thermo Scientific, 25mg/LKANAMYCIN,25mg/L GENTAMYCIN,50mg/L KANAMYCIN), single clones were picked up and cultured overnight at 28℃on 4mL RKG liquid medium (tryptone 10g, yeast extract 5g, naCl10 g), and tobacco leaves were transformed according to the tobacco transient transformation step. And marking. After 2 days, luciferase substrate (D-Luciferin (potassium salt), APExBIO) was injected into the transient transformation site using a 1mL needleless syringe, the leaves were placed in an exposure apparatus for luminescence signal detection and pseudo-color was added (FIG. 1, D).

Example 2 amino acid residues 49 and 50 of NIB1 are important for the NIB1 to function

Protein structure simulation and key amino acid site mutation

The possible protein structure of NIB1 was obtained by molecular modeling of NIB1 with reference to the crystal structure of tobacco RNA binding protein NbGRP, and its binding site for 2',3' -cAMP and 2',3' -cGMP was obtained, including amino acid residues at positions 49 and 50 in the RRM domain (FIGS. 2, A-C). Site-directed mutagenesis was performed on these sites (mutation method is substitution mutation, the gene was segmented at the mutation site, mutant nucleobases were introduced into the primer, the vector was constructed by means of multi-segment homologous recombination), and micro thermophoresis (MST) was performed to examine its ability to bind small molecules, which revealed that NIB1 had the ability to bind 2',3' -cAMP, whereas NIB1 ^R49A had the ability to bind NIB1 ^G50A diminished or even disappeared (FIG. 2, D).

Observation of the formation of coagulum of NIB1 in plant nuclei by fluorescence confocal microscopy

Cutting tobacco plant leaves with a certain size, inversely placing the tobacco plant leaves on a glass slide, adding a drop of ddH ₂ O, lightly covering the glass slide, absorbing excessive water, inversely placing the glass slide on a stage, firstly using a low-power lens to find a target view, moving to the center of the view, switching to a high-power lens, selecting corresponding fluorescence excitation wavelength and receiving wavelength, and adjusting a Z axis by adjusting excitation intensity and related parameters to acquire images. In the shooting process, overexposure is avoided, YFP used in the study is acquired through a GFP channel, excitation wavelength is 488nm, receiving wavelength is 498-550 nm, and acquired images are added with a scale and quantized by using Image J software. Microscopic observation of NIB1-YFP tobacco leaves revealed that NIB1-YFP formed bright spots in the nuclei, whereas NIB1 ^R49A and NIB1 ^G50A were not (FIG. 2, E-F).

Tobacco expression protein

The constructed vector was transformed into Agrobacterium GV3101 competent cells, and the plates were cultured upside down at 28℃for 2 days. After two days, the single clones on the plates were picked separately and transferred to 4mL RKG liquid medium, shake-cultured overnight at 28℃and a small amount of agrobacteria were 1:100 amplified into fresh RKG medium, shake-cultured overnight at 28 ℃. Centrifuging for 5 minutes at 4 ℃ and 5000g, pouring out excess LB, re-suspending thalli by using tobacco transient re-suspension to enable the bacterial liquid OD600 to be 0.6, standing for 2-3 hours at room temperature, and preparing for injection. Two days later, the results of tobacco injections with NIB1-YFP, NIB1 ^R49A and NIB1 ^G50A, respectively, showed that the sites of NIB1-YFP injection caused plant cell death, whereas NIB1 ^R49A and NIB1 ^G50A were not (FIG. 2, G).

D, experiment of feeding cotton bollworms

After a large amount of NIB1-YFP, NIB1 ^R49A and NIB1 ^G50A are injected, tobacco leaves are cut into the same size, leaves with the same position and the same size are taken out in a 6-hole culture dish, two-year cotton bollworms with the same size (purchased from Henan cloud biological company) are selected, placed in the culture dish and weighed, during the weighing process, fresh plant leaves with the same size are placed every other day, after three days of full feeding, weighing is carried out, and the obtained data is subtracted by the data fed on the first day, namely the weight gain of the cotton bollworms. The results showed that NIB1 increased the resistance of tobacco to cotton bollworms, whereas NIB1 ^R49A and NIB1 ^G50A were not (FIG. 2, H).

Agrobacterium infection buffer 10mM MgCl ₂, 10mM MES, 150. Mu.M acetosyringone, KOH adjusted to pH 5.8.

EXAMPLE 3 NIB1 accelerates NVR1 Activity

A in vitro detection of the Effect of NIB1 on NVR1 Activity

In vitro, using DNA as template and Cy5-UTP as substrate, performing transcription, removing DNA template to obtain Cy 5-labeled RNA, then reacting with substrate 1:100 according to NVR1 and NVR1 of 1:1 of NIB1, at 25deg.C for 25min, adding high salt buffer to terminate enzyme activity reaction, purifying with phenol-chloroform and isopropanol, re-dissolving, and adding 2× TBE urea loading buffer (0.5×TBE,6M urea, 0.01% bromophenol blue, 15%)PM 400), 95℃for 3min, immediately on ice, urea polyacrylamide gel electrophoresis, scanning detection signal using TYPHOON5, and quantification of the bands using Image J. As shown in FIGS. 3, A and E, NIB1 significantly promoted NVR1 catalytic activity, whereas NIB1 ^ΔIDR was not.

B, high performance liquid chromatography for detecting catalytic products

The small molecule separation uses a chromatographic column XSelect HSS T3 XP (Waters), wherein the aqueous phase is water containing 2mM amine acetate and the organic phase is methanol containing 2mM amine acetate. The separation conditions were as follows:

The results are shown in FIG. 3B, C, F, G, I and J, which show that NIB1 promotes the production of more active small molecules 2',3' -cAMP and 2',3' -cGMP, whereas NIB1 ^ΔIDR and NIB1 ^R49A do not have this capability and NIB1 ^G50A.

Co-transformation in tobacco cells to verify the effect of NIB1 on NVR1 induced necrosis

The constructed vector NVR1-YFP and NIB1-YFP (YFP) were co-transformed into competent cells of Agrobacterium GV3101 and the plates were cultured for 2 days in an inverted position at 28 ℃. After two days, the single clones on the plates were picked separately and transferred to 4mL RKG liquid medium, shake-cultured overnight at 28℃and a small amount of agrobacteria were 1:100 amplified into fresh RKG medium, shake-cultured overnight at 28 ℃. Centrifuging for 5 minutes at 4 ℃ and 5000g, pouring out excess LB, re-suspending thalli by using tobacco transient heavy suspension to enable the bacterial liquid OD ₆₀₀ to be 1, standing for 2-3 hours at room temperature, uniformly mixing every two bacteria according to equal volume, and preparing for injection. The results showed that NIB1 ^ΔIDR and NIB1 ^R49A and NIB1 ^G50A had reduced ability to accelerate necrosis of NVR1 compared to NIB1 (fig. 3, d, H, k and H).

EXAMPLE 4 NIB1 enables recruitment of EDS1 to the aggregate

BiFC verifies NIB1 interaction with EDS1

Proteins EDS1 and NIB1 for verifying interaction were ligated to MSH21-35S-cGFP vector and MSH22-35S-nGFP vector, respectively. After the sequence to be tested is correct, the two vectors are respectively transformed into agrobacterium GV3101, after the two vectors are cultured on a RKG-containing solid medium for two days at 28 ℃, single clone is selected and cultured on 4mL RKG liquid medium at 28 ℃ overnight, tobacco leaves are transformed according to the tobacco transient transformation step, and the tobacco leaves are marked. After 2 days, observations were made under a fluorescence confocal microscope and images were taken, which showed that NIB1 interacted with EDS1 (FIG. 4, D).

Co-localization observations of NIB1 recruitment of EDS1 into cell nucleus aggregates

NIB1 and EDS1 were respectively ligated to vectors with YFP and mCherry, after correct sequencing, the two vectors were transformed into Agrobacterium GV3101 respectively, after two days of cultivation on RKG-containing solid medium at 28℃the individual clones were picked up in 4mL RKG liquid medium, after overnight cultivation at 28℃the tobacco leaves were transformed at 1:1 and marked. After 2 days, microscopic observations were made, which indicated that NIB1-YFP was able to recruit EDS1-mCherry to bright bodies in the nucleus (FIG. 4, E).

EXAMPLE 5 NIB1 upregulates plant pest control ability

In vitro experiments on Pyricularia oryzae

The stored TH12 Pyricularia oryzae strain (laboratory preservation) was inoculated into CM solid medium, cultured at 28℃for 10 days, spores were eluted with sterilized water, and then counted by a hemocytometer to adjust the spore concentration to 6X 10 ⁵ spores/mL. Taking rice leaves in the tillering stage, cutting young rice leaves into small sections with the length of about 10cm, putting the leaves into an agar solid culture dish, paying attention to moisture preservation at two ends, preventing the leaves from losing moisture, rubbing and scratching the surfaces of the leaves by using a 1mL syringe needle, then dripping 10 mu L of spore liquid, and then putting into an inoculation box. As shown in FIGS. 5A-C, NIB1 positively regulates plant resistance to rice blast.

CM medium formulation ：20×Nitrate salts(50mL),1000×Trace elements(1mL),1000×Vitamin solution(1mL),D-glucose 10g,Peptone 2g,Yease extract 1g,Casamino acid 1g.10M NaOH was adjusted to ph=6.5, ddH ₂ O was added to 1000ml and autoclaved at 115 ℃ for 20min. If solid medium is needed, 15g of agar powder is added per liter.

NIB1-OE transgenic plants are provided by Wuhan Boehringer and are responsible for experimental processes such as vector, agrobacterium, rice strain, transformation, culture, passage, etc.

B test experiment of cnaphalocrocis medinalis

The method comprises the steps of taking rice leaves in the tillering stage, cutting young rice leaves into small sections with the length of about 10cm, placing the small sections in culture dishes paved with soaked gauze, paving about 7 leaves on each culture dish, placing three-instar nymphs of cnaphalocrocis medinalis (collected in Songjiang farm in Shanghai city) on the rice leaves, placing paper blocks on two sides of the rice leaves, supplementing water every day, and preventing rice from drying up. Three days later, the weights of the worms were weighed, and as shown in D-F of FIG. 5, the weights of the insects of plants feeding NIB1-KO were significantly increased compared to the control group and NIB1-OE (over-expressing NIB 1).

TABLE 1 primers used in this study

Sequences herein

SEQ ID NO. 1-NIB1 Gene nucleotide sequence ATGGCGGCGCCGGATGTCGAGTACCGCTGCTTCGTCGGCGGCCTCGCCTGGGCCACCGACGACCGCTCCCTCGAGGCCGCCTTCTCCACCTACGGCGAGATCCTCGACTCCAAGATCATCAACGACAGGGAGACGGGGAGGTCACGTGGGTTTGGCTTCGTCACCTTCTCCTCCGAGCAGTCGATGCGCGACGCCATCGAGGGCATGAACGGCAAGGAGCTCGACGGCCGCAACATCACCGTCAATGAGGCCCAGTCCCGCCGCTCCGGCGGCGGAGGCGGGGGCTACGGCGGCGGCGGTGGCGGCTACGGCGGCGGTCGTGGAGGCGGCGGCTACGGAGGAGGTGGCGGCGGCGGCTACGGGCGCCGTGAGGGCGGCTACGGCGGCGACTCCGGCGGGAACTGGAGGAAC

SEQ ID NO. 2-NIB1 Gene amino acid sequence

MAAPDVEYRCFVGGLAWATDDRSLEAAFSTYGEILDSKIINDRETGRSRGFGFVTFSSEQSMRDAIEGMNGKELDGRNITVNEAQSRRSGGGGGGYGGGGGGYGGGRGGGGYGGGGGGGYGRREGGYGGDSGGNWRN.

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, wherein 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, wherein the polypeptide encoded by the NIB1 gene is selected from the group consisting of:

(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, wherein 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' and/or 3' ends 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.