Rice disease resistance genes and uses thereof

Abstract

The present application relates to the field of biotechnology, in particular to the use of Flotillin1 gene and Importinα4 gene, and the use of molecules inhibiting the transcription or translation of Flotillin1 gene and Importinα4 gene in the preparation of a kit. The present application also relates to a method for obtaining a plant capable of resisting pathogen.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to the use of the rice Flotillin1 and Importinα4 genes, and the use of molecules that inhibit the transcription or translation of the Flotillin1 and Importinα4 genes in the preparation of a kit. This application also relates to a method for obtaining plants capable of resisting pathogens. Background Technology

[0002] Plant viral infections have become the second leading cause of agricultural disease, causing enormous economic losses globally each year. Rice viruses are a significant group of plant viruses, seriously threatening rice yields in East Asia, leading to yield reductions of up to 16% or even total crop failure. Recent outbreaks have primarily involved rice stripe virus (RSV), southern rice black-streaked dwarf virus (SRBSDV), rice black-streaked dwarf virus (RBSDV), rice rice toothed dwarf virus (RRSV), and rice grassy dwarf virus (RGSV). Therefore, blocking viral transmission within plant hosts and cultivating broad-spectrum resistant varieties are crucial approaches to combating viral diseases.

[0003] Rice stripe virus (RSV) is a single-stranded RNA virus with a genome consisting of four RNA strands (RNA1, RNA2, RNA3, and RNA4), encoding seven proteins (RdRp, NS2, NSvc2, NS3, NP, SP, and NSvc4). Current research on RSV and its interaction with the rice host focuses on the interaction between RSV-encoded proteins and rice host factors, affecting viral replication or influencing gene silencing, immunity, and autophagy pathways in plant host cells (Fu et al., 2018; Zheng et al., 2017; Zhao et al., 2016; Kong et al., 2014). However, the receptor for RSV in the rice host remains unclear.

[0004] In conclusion, plant virus infections pose a serious threat to global economic and food security, but research on blocking virus transmission is relatively scarce. There is a need to develop a disease-resistant gene to combat viral infections in rice. Summary of the Invention

[0005] Through extensive experimentation and repeated exploration, the inventors of this application discovered that the Flotillin1 gene and Importinα4 in plants play a negative regulatory role in plant pathogen resistance. Therefore, they prepared rice mutants with Flotillin1 and Importinα4 knocked out, and unexpectedly found that these mutants significantly inhibited the spread of RSV in rice and significantly reduced the incidence of RSV in rice. Furthermore, these mutants had no effect on rice plant height, thousand-grain weight, or germination rate. Therefore, mutant plants with Flotillin1 and Importinα4 gene knockout have good application potential in plant disease resistance.

[0006] Therefore, in a first aspect, this application provides a method for obtaining a plant capable of resisting pathogens, the method comprising: reducing or inhibiting the transcription or translation of the Flotillin1 gene and the Importin α4 gene in the plant, thereby reducing or inhibiting the expression levels of the proteins encoded by the Flotillin1 gene and the Importin α4 gene, respectively, in the plant.

[0007] In some embodiments, the plant is a grass (Poaceae). In some embodiments, the plant is selected from wheat, barley, corn, rice, and sorghum.

[0008] In some embodiments, the pathogen is a virus. In some embodiments, the pathogen is rice stripe virus.

[0009] In some embodiments, the amino acid sequence of the protein encoded by the Importinα4 gene is shown in SEQ ID NO:3.

[0010] In some embodiments, the amino acid sequence of the protein encoded by the Flotillin1 gene is shown in SEQ ID NO:1.

[0011] In some embodiments, the reduction or suppression of the expression of the plant Flotillin1 and Importin α4 genes is achieved by any of the following methods: substitution, deletion, or addition of one or more nucleotides (e.g., substitution, deletion, or addition of 1, 2, or 3 nucleotides), site-specific mutagenesis, ethyl mesylate mutagenesis, directed induction of local genomic mutations, or gene editing (e.g., gene knockout guided by sgRNA).

[0012] In some implementations, sgRNAs targeting the Flotillin1 and Importin α4 genes are designed to guide Cas9 to edit the target sites.

[0013] In some embodiments, when several (e.g., 1, 2, 3, 4) copies of the Flotillin1 gene and the Importin α4 gene are present in the plant cell, each copy of the Flotillin1 gene and the Importin α4 gene is made defective.

[0014] In a specific embodiment of the present invention, sgRNA sequences targeting the rice Flotillin1 gene and Importin α4 gene were designed, and corresponding vectors containing sgRNA and Cas9 protein were constructed. Using Agrobacterium as a gene manipulation tool, the sgRNA sequences targeting the rice Flotillin1 gene and Importin α4 gene were efficiently introduced into the rice cell genome using the vectors, reducing the expression levels of the rice Flotillin1 gene and Importin α4 gene, and significantly downregulating the expression of Flotillin1 gene and Importin α4 at both the mRNA and protein levels.

[0015] Therefore, in some implementations, the method is achieved through the following steps (a) to (f):

[0016] (a) Construct a vector containing sgRNA1 and sgRNA2, wherein sgRNA1 is capable of targeting the Flotillin1 gene or a fragment thereof, and sgRNA2 is capable of targeting the Importin α4 gene or a fragment thereof;

[0017] (b) Transform the vector into Agrobacterium;

[0018] (c) Infecting plant cells with the Agrobacterium;

[0019] (d) Optionally, select plant cells with defective Flotillin1 and Importin α4 genes;

[0020] (e) Generate a plant from the plant cells of step (c) or (d);

[0021] (f) Optionally, plants with defective Flotillin1 and Importin α4 genes are screened to obtain plants that are resistant to pathogens.

[0022] In some embodiments, sgRNA1 and sgRNA2 are constructed in the same vector or different vectors. In some embodiments, sgRNA1 and sgRNA2 are constructed in the pYLCRISPR / Cas9Pμbi-H vector.

[0023] In some embodiments, the Agrobacterium is Agrobacterium EHA105.

[0024] In some embodiments, in step (f), RNA is extracted from the plant, the RNA is reverse transcribed into cDNA, and nucleotide fragments targeting sgRNA1 and sgRNA2 contained in the cDNA are amplified using primers for screening; or, genomic DNA of the plant is extracted, and nucleotide fragments targeting sgRNA1 and sgRNA2 contained in the DNA are amplified using primers for screening.

[0025] In some embodiments, the nucleotide sequence of the sgRNA1 is SEQ ID NO:5 or SEQ ID NO:6.

[0026] In some embodiments, the nucleotide sequence of the sgRNA2 is SEQ ID NO:7 or SEQ ID NO:8.

[0027] In a second aspect, this application provides the use of a molecule that specifically inhibits the transcription or translation of the Flotillin1 gene and the Importinα4 gene, or specifically inhibits the expression levels of proteins encoded by the Flotillin1 gene and the Importinα4 gene, in a preparation kit for obtaining plants resistant to pathogens, or for improving the ability of plants to resist pathogens.

[0028] In one embodiment of the present invention, the molecules mentioned include, but are not limited to, nucleic acid molecules, carbohydrates, lipids, small molecule chemicals, antibody drugs, polypeptides, proteins, or interfering lentiviruses.

[0029] In one embodiment of the present invention, the nucleic acid molecule includes, but is not limited to: antisense oligonucleotides, double-stranded RNA (dsRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), or guide RNA (sgRNA).

[0030] In some embodiments, the nucleic acid molecule comprises at least two, at least three, at least four, at least five, or at least six sgRNAs.

[0031] In some embodiments, the nucleic acid molecule comprises sgRNA1 and sgRNA2, wherein sgRNA1 is capable of targeting the Flotillin1 gene or a fragment thereof, and sgRNA2 is capable of targeting the Importin α4 gene or a fragment thereof.

[0032] In some embodiments, the nucleotide sequence of the sgRNA1 is SEQ ID NO:5 or SEQ ID NO:6.

[0033] In some embodiments, the nucleotide sequence of the sgRNA2 is SEQ ID NO:7 or SEQ ID NO:8.

[0034] In some embodiments, the kit further comprises: a vector, Agrobacterium, a culture medium and / or reagents for culturing plant cells or tissues, reagents for extracting plant DNA or RNA, a primer set for amplifying fragments targeting sgRNA1 and sgRNA2, respectively, or any combination thereof.

[0035] In some embodiments, the vector is pYLCRISPR / Cas9Pμbi-H.

[0036] In some embodiments, sgRNA1 and sgRNA2 may or may not be contained in the vector.

[0037] In some embodiments, the Agrobacterium is Agrobacterium EHA105.

[0038] In some embodiments, the plant is a grass (Poaceae). In some embodiments, the plant is selected from wheat, barley, corn, rice, and sorghum.

[0039] In some embodiments, the pathogen is a virus. In some embodiments, the pathogen is rice stripe virus.

[0040] In some embodiments, the amino acid sequence of the protein encoded by the Importinα4 gene is shown in SEQ ID NO:3.

[0041] In some embodiments, the amino acid sequence of the protein encoded by the Flotillin1 gene is shown in SEQ ID NO:1.

[0042] In a third aspect, this application provides a kit comprising any one or more of the following (1) to (6):

[0043] (1) Nucleic acid molecules that specifically inhibit the transcription or translation of the Flotillin1 gene and the Importinα4 gene or specifically inhibit the expression level of the proteins encoded by the Flotillin1 gene and the Importinα4 gene, respectively.

[0044] (2) A carrier containing the nucleic acid molecule;

[0045] (3) Agrobacterium containing the vector;

[0046] (4) Culture media and / or reagents used for culturing plant cells or tissues;

[0047] (5) Reagents used to extract plant DNA or RNA;

[0048] (6) Primer set for amplifying nucleotide fragments containing the Flotillin1 gene and the Importin α4 gene.

[0049] In some implementations, the kit comprises all combinations of (1) to (6).

[0050] In some embodiments, the nucleic acid molecule is selected from antisense oligonucleotides, dsRNA, siRNA, shRNA, or sgRNA.

[0051] In some embodiments, the nucleic acid molecule comprises sgRNA1 and sgRNA2, wherein sgRNA1 is capable of targeting the Flotillin1 gene or a fragment thereof, and sgRNA2 is capable of targeting the Importin α4 gene or a fragment thereof.

[0052] In some embodiments, the kit is used to obtain plants that are resistant to pathogens, or to enhance the ability of plants to resist pathogens.

[0053] In some embodiments, the plant is a grass (Poaceae). In some embodiments, the plant is selected from wheat, barley, corn, rice, and sorghum.

[0054] In some embodiments, the pathogen is a virus. In some embodiments, the pathogen is rice stripe virus.

[0055] In some embodiments, the amino acid sequence of the protein encoded by the Importinα4 gene is shown in SEQ ID NO:3.

[0056] In some embodiments, the amino acid sequence of the protein encoded by the Flotillin1 gene is shown in SEQ ID NO:1.

[0057] In some embodiments, the nucleotide sequence of the sgRNA1 is SEQ ID NO:5 or SEQ ID NO:6.

[0058] In some embodiments, the nucleotide sequence of the sgRNA2 is SEQ ID NO:7 or SEQ ID NO:8.

[0059] In some embodiments, the vector is pYLCRISPR / Cas9Pμbi-H.

[0060] In some embodiments, the Agrobacterium is Agrobacterium EHA105.

[0061] Terminology Definition

[0062] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational steps used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all conventional steps widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0063] As used herein, the term “defective” refers to a reduction in the level of a protein encoded by a gene in a cell or plant compared to that of a wild-type plant, by approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (i.e., complete suppression of the protein level).

[0064] As used herein, the term "protein encoded by the Flotillin1 gene" refers to the naturally occurring, biologically active Flotillin1 protein. The Flotillin1 protein is found in a variety of grasses, and its amino acid sequence is readily available from various public databases (e.g., GenBank). In some embodiments, the amino acid sequence of the protein encoded by the Flotillin1 gene is as shown in SEQ ID NO:1.

[0065] As used herein, the term "protein encoded by the Importin α4 gene" refers to the naturally occurring, biologically active Importin α4 protein. Importin α4 protein is found in a variety of grasses, and its amino acid sequence is readily available from various public databases (e.g., GenBank). In some embodiments, the amino acid sequence of the protein encoded by the Importin α4 gene is shown in SEQ ID NO:3.

[0066] As used herein, the term "Flotillin1 gene" refers to any nucleic acid that encodes the Flotillin1 protein, including DNA (e.g., genomic DNA) and RNA (e.g., mRNA). In some embodiments, the nucleotide sequence of the Flotillin1 gene is as shown in SEQ ID NO:2.

[0067] As used herein, the term "Importin α4 gene" refers to any nucleic acid encoding the Importin α4 protein, including DNA (e.g., genomic DNA) and RNA (e.g., mRNA). In some embodiments, the nucleotide sequence of the Importin α4 gene is as shown in SEQ ID NO:4.

[0068] As used herein, the term "pathogen" refers to all organisms capable of causing disease in plants, including fungi, nematodes, bacteria, and viruses. In some embodiments, the pathogen is rice stripe virus.

[0069] As used herein, the term "plant resistant to pathogens" means that a plant is able to resist the infection and / or spread of the pathogen to a certain extent, specifically, that after the pathogen infects the plant, the plant exhibits reduced disease incidence compared to wild-type plants.

[0070] As is known to those skilled in the art, codons exhibit degeneracy. That is, during protein translation, each amino acid can correspond to one or more codons, for example, up to six codons. Different species show significant differences in their use of degenerate codons encoding a particular amino acid, exhibiting different preferences. This preference phenomenon is known as "codon bias." Therefore, as used herein, the term "codon bias" refers to the situation where a species prefers to use certain specific codons to encode amino acids. Optimizing the sequence of nucleic acid molecules based on codon bias can be particularly advantageous in certain situations, for example, it may help improve the expression level of the protein encoded by the nucleic acid molecule.

[0071] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids (e.g., naked plasmids); phage particles; Cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and viral vectors. Viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (e.g., SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site.

[0072] As used herein, the term "sgRNA (small guide RNA)" refers to a guide RNA used to target a specific nucleic acid, under the guidance of the sgRNA, allowing the Cas9 protein to perform site-specific editing. Typically, in CRISPR / Cas9 genome editing technology, sgRNAs are designed to target specific sites on the target nucleic acid, guiding the Cas9 nuclease to do so. Not every sgRNA has the same cleavage efficiency. Given this inconsistency, it is necessary to screen multiple sgRNAs to find the one with the highest cleavage efficiency. In some embodiments, the nucleotide sequence of the sgRNA targeting the Flotillin1 gene is SEQ ID NO:5 or SEQ ID NO:6. In some embodiments, the nucleotide sequence of the sgRNA targeting the Importin α4 gene is SEQ ID NO:7 or SEQ ID NO:8.

[0073] Beneficial effects of the invention

[0074] The applicant prepared rice mutants with single knockouts of Importinα4 or Flotillin1. Experimental tests showed that the single knockout mutants were not very effective. Furthermore, the applicant unexpectedly discovered that rice mutants with simultaneous knockouts of Flotillin1 and Importinα4 significantly inhibited the spread of RSV in rice (at least 50% inhibition compared to WT) and also significantly reduced the incidence of RSV in rice (at least 40% reduction compared to WT). Moreover, this mutant had no effect on rice plant height, thousand-grain weight, or germination rate. Therefore, this mutant and its knockout combinations (i.e., knockout of the Flotillin1 and Importinα4 genes) have good application potential in plant disease resistance.

[0075] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0076] Figure 1 The results of validation at the transcriptional and protein levels of the rice Flotillin1 mutant are shown, among which, Figure 1 In this context, A represents the expression level of Flotillin1 transcription in wild-type rice (WT) and the Flotillin1 mutant. Figure 1In this context, B represents the expression level of Flotillin1 protein in wild-type rice (WT) and the Flotillin1 mutant. Values ​​are expressed as mean ± standard error, and different lowercase letters indicate significant differences in gene expression between different treatments (P < 0.05).

[0077] Figure 2 The results show the effects of Flotillin1 knockout on rice plant height, thousand-grain weight, and germination rate. Figure 2 In A in 2 and B in 2, the changes in rice plant height after knocking out Flotillin1 are indicated by a scale bar of 10 cm. Figure 2 In the figures C and D, 2 represents the change in thousand-grain weight of rice after knocking out Flotillin1. The scale bar is 5 cm. Values ​​are expressed as mean ± standard error. Different lowercase letters indicate significant differences in gene expression between different treatments (P < 0.05).

[0078] Figure 3 The effect of Flotillin1 knockout on RSV diffusion rate in rice was shown, where, Figure 3 In A and B in 3, the diffusion rate of RSV in epidermal cells, fibrous tissue, mesophyll cells, bundle sheath cells, sieve tubes and companion cells in Flotillin1 mutant 12 h after RSV infection of inoculated leaves is represented by a scale bar of 20 nm. Figure 3 In C and D in 3, the diffusion rate of RSV in epidermal cells, fibrous tissue, mesophyll cells, bundle sheath cells, sieve tubes and companion cells 1 day after RSV infection of the Flotillin1 mutant is indicated by a scale bar of 20 nm.

[0079] Epi, epidermal cells, Fib, fibrous tissue, Mes, mesophyll cells, Bs, bundle sheath cells, SE, sieve tubes, CC, companion cells. Blue fluorescence indicates plasmodesmata staining signal, and red fluorescence indicates RSV NP signal. Values ​​are expressed as mean ± standard error, and different lowercase letters indicate significant differences in gene expression between different treatments (P < 0.05).

[0080] Figure 4 The effect of Flotillin1 knockout on RSV NP expression levels in rice was shown. Figure 4 In the table, A, B, and C represent the changes in NP expression levels in rice systems infected with RSV 1d, 4d, and 7d after infection, respectively. Values ​​are expressed as mean ± standard error, and different lowercase letters indicate significant differences in gene expression between different treatments (P < 0.05).

[0081] Figure 5 The effects of Flotillin1 knockout on rice disease incidence were shown, among which, Figure 5In this context, A represents the effect of RSV infection on rice disease incidence in the Flotillin1 mutant when the insect population density is 10 individuals. Figure 5 In the figure, B represents the effect of RSV infection on the disease incidence of Flotillin1 mutant rice when the insect population density is 2. Values ​​are expressed as mean ± standard error. An asterisk indicates a significant difference between treatments, *P<0.05, **P<0.01, and different lowercase letters indicate significant differences in gene expression between different treatments (P<0.05).

[0082] Figure 6 The effects of knocking out Importin α4 on rice plant height and thousand-grain weight were shown. Figure 6 In the figure, A and B represent the changes in rice plant height after knocking out Importinα4, with a scale bar of 10 cm. Figure 6 In the figure, C represents the change in thousand-grain weight of rice after knocking out Importinα4. Values ​​are expressed as mean ± standard error, and different lowercase letters indicate significant differences in gene expression between different treatments (P<0.05).

[0083] Figure 7 The effect of knocking out Importinα4 on the expression level of RSV NP in rice was shown. Figure 7 A, B, and C represent the changes in NP expression levels in rice systematic leaves 1, 4, and 7 days after RSV infection in the Importinα4 mutant, respectively. Values ​​are expressed as mean ± standard error, and different lowercase letters indicate significant differences in gene expression between different treatments (P < 0.05).

[0084] Figure 8 The effects of knocking out Importin α4 on rice disease incidence were shown. Figure 8 In the figure, A represents the effect of RSV infection on rice disease incidence in the Importin α4 mutant when the insect population density is 10. Figure 8 In the figure, B represents the effect of RSV infection on the disease incidence of Importin α4 mutant rice when the insect population density is 2. Values ​​are expressed as mean ± standard error. An asterisk indicates a significant difference between treatments, *P<0.05, **P<0.01, and different lowercase letters indicate significant differences in gene expression between different treatments (P<0.05).

[0085] Figure 9 The effects of knocking out Flotillin1-Importinα4 on rice plant height and thousand-grain weight were shown. Figure 9 In the figure, A and B represent the changes in rice plant height after knocking out Flotillin1-Importinα4, with a scale bar of 10 cm. Figure 9In the figure, C represents the change in thousand-grain weight of rice after knocking out Flotillin1-Importinα4. Values ​​are expressed as mean ± standard error, and different lowercase letters indicate significant differences in gene expression between different treatments (P<0.05).

[0086] Figure 10 The effect of knocking out Flotillin1-Importinα4 on the expression level of RSV NP in rice was shown. Figure 10 In the table, A, B, and C represent the changes in NP expression levels in rice systematic leaves 1, 4, and 7 days after RSV infection in the Flotillin1-Importinα4 mutant. Values ​​are expressed as mean ± standard error, and different lowercase letters indicate significant differences in gene expression between different treatments (P < 0.05).

[0087] Figure 11 The effects of knocking out Flotillin1-Importinα4 on rice disease incidence were shown. Figure 11 In the figure, A represents the effect of RSV infection on rice disease incidence in the Flotillin1-Importin α4 mutant when the insect population density is 10. Figure 11 In the figure, B represents the effect of RSV infection on the disease incidence of Flotillin1-Importin α4 mutant rice when the insect population density is 2. Values ​​are expressed as mean ± standard error. An asterisk indicates a significant difference between treatments, *P<0.05, **P<0.01, and different lowercase letters indicate significant differences in gene expression between different treatments (P<0.05).

[0088] Sequence information

[0089] Information on some of the sequences involved in this invention is provided in Table 1 below.

[0090] Table 1: Sequence Description

[0091]

[0092]

[0093] Detailed Implementation

[0094] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0095] Unless otherwise specified, the experiments and methods described in the embodiments are performed in accordance with conventional methods well known in the art and described in various references. For example, conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in this invention can be found in Sambrook, Fritsch, and Maniatis, *Molecular Cloning: A Laboratory Manual*, 2nd edition (1989); *Current Protocols in Molecular Biology* (edited by FM. Ausubel et al., (1987)); the *Methods in Enzymology* series (academic publishing company): *PCR 2: A PRACTICAL APPROACH* (edited by MJ. MacPherson, BD. Hames, and GR. Taylor, (1995)); and *Animal Cell Culture*. CELLCULTURE (edited by R.R. Freshney (1987)).

[0096] Furthermore, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the examples are described by way of illustration and are not intended to limit the scope of protection claimed by the invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0097] Example 1. Test insects and test plants

[0098] Test insects: The poisoned and non-poisonous planthopper strains (Laodelphax striatellμs) used in this study were mainly collected from Hai'an, Jiangsu Province, and domesticated over a long period. The planthoppers were reared in glass bottles containing 2-3 cm rice seedlings, sealed with nylon mesh. The rearing temperature was maintained at approximately 25 degrees Celsius, with a photoperiod of 16h:8h (light:dark). To ensure sufficient nutrition, the rice seedlings were replaced weekly. Poisoned and non-poisonous planthopper strains were reared in different greenhouses under identical environmental conditions, and virus load was detected and screened every three months using dot-linked enzyme-linked immunosorbent assay (ELISA).

[0099] Screening for virus-carrying planthoppers: To obtain a planthopper strain with a high virus-carrying rate, we regularly screen virus-carrying populations. Forty-five pregnant female planthoppers are collected and reared in separate bottles. After hatching, five larvae are randomly selected from each bottle, and the virus load in the larvae is tested using a dot-matrix enzyme-linked immunosorbent assay (ELISA). The offspring of all tested larvae, all of whom are virus-carrying female planthoppers, are then mixed and propagated to form a virus-carrying population. The specific steps for the dot-matrix ELISA procedure are as follows:

[0100] (1) Place the planthopper to be tested into a 0.2 mL PCR tube, one planthopper per tube, add 5 μL of 0.05 M carbonate buffer, and grind it using a pipette tip that has been treated at the tip.

[0101] (2) Take 3 μL of the ground sample onto the nitrocellulose membrane, place it in an incubation box, and air dry it in a fume hood.

[0102] (3) Blocking: Add 10 mL of 1% skim milk (prepared with 1xPBST buffer) to the incubator containing the dried nitrocellulose membrane, place it on a shaker, and block for 30 min at room temperature.

[0103] (4) Incubation of primary antibody: Pour out the blocking solution, dilute RSV NP monoclonal antibody with 1% skim milk at a ratio of 1:5000 (Zhao, et al., 2016), add 10 mL to the incubation box, place on a shaker, and incubate at room temperature for 2 h.

[0104] (5) Washing the primary antibody: Pour out the primary antibody dilution solution, add 1x PBST buffer, and wash on a shaker for 15 min. Repeat 2-3 times.

[0105] (6) Incubation of secondary antibody: Dilute goat anti-mouse secondary antibody (Kangwei Century, CW0102S) containing horseradish peroxidase (HRP) with 1% skim milk at a ratio of 1:10000, add 10 mL to the incubation box, place on a shaker, and incubate at room temperature for 1 h.

[0106] (7) Washing the secondary antibody: Pour out the secondary antibody dilution solution, add 1x PBST buffer, and wash on a shaker for 15 minutes. Repeat 2-3 times.

[0107] (8) Place the cleaned membrane into the color development solution (10 mL of 2 mM PBS solution, 2 mL of anhydrous ethanol, 6 μg of 4-chloro-1-naphthol, and 7 μL of 30% H2O2) and place it on a shaker for 2 h at room temperature.

[0108] (9) After color development, rinse three times with tap water and air dry at room temperature. If blue-purple spots appear, the sample is toxic.

[0109] Test plants: Japanese rice (Oryza sativa Japonica Groμp) were cultured in an artificial culture rack at a temperature of 26℃ / 24℃, a photoperiod of L:D = 14:10, and a relative humidity of 70%. The rice was used in experiments after 45 days of culture.

[0110] Example 2. Preparation of rice mutants

[0111] 1. Rice Flotillin1 mutant

[0112] (1) Vector construction: Two Flotillin1 targeting sgRNAs (the amino acid sequence is shown in SEQ ID NO:1 and the nucleotide sequence is shown in SEQ ID NO:2) were designed and sequentially ligated into the CRISPR / Cas9 binary vector pYLCRISPR / Cas9Pμbi-H (Wuhan Boyuan Biotechnology).

[0113] Table 2. sgRNA sequence of Flotillin1

[0114]

[0115] (2) The target vector was transformed into Agrobacterium EHA105 and Agrobacterium-mediated transformation into embryogenic callus of Nipponbare rice. At least three homozygous mutants with Flotillin1 gene knockout were identified.

[0116] (3) Molecular identification of Flotillin1 mutant rice: DNA was extracted from transgenic plants, primers were designed at both ends of the targeting sgRNAs, PCR was performed to amplify the fragment containing the targeting region, and sequencing analysis was performed to analyze the targeting effect. The sequences of primers F1 and R1 for detecting the targeting effect are as follows: forward primer F1:5 (nucleotide sequence as shown in SEQ ID NO:15), reverse primer R1:5 (nucleotide sequence as shown in SEQ ID NO:16).

[0117] 2. Rice Importin α4 mutant

[0118] (1) Vector construction: Two target sgRNAs of Importin α4 (the amino acid sequence is shown in SEQ ID NO:3 and the nucleotide sequence is shown in SEQ ID NO:4) were designed (Table 3) and sequentially ligated into the CRISPR / Cas9 binary vector pYLCRISPR / Cas9Pμbi-H (Wuhan Boyuan Biotechnology).

[0119] Table 3. sgRNA sequence of OsImportin α4

[0120]

[0121] (2) The target vector was transformed into Agrobacterium EHA105 and Agrobacterium-mediated transformation into embryogenic callus of Nipponbare rice. At least three homozygous Importin α4 gene knockout mutants were identified.

[0122] (3) Molecular identification of Importin α4 mutant rice: DNA was extracted from transgenic plants, primers were designed at both ends of the target sgRNAs, PCR was performed to amplify the fragment containing the target region, and sequencing analysis was performed to analyze the targeting effect. The sequences of primers F1 and R1 for detecting the targeting effect are as follows: forward primer F1:6 (nucleotide sequence as shown in SEQ ID NO:19), reverse primer R1:6 (nucleotide sequence as shown in SEQ ID NO:20).

[0123] 3. Rice Importin α4-Flotillin1 mutant

[0124] (1) Vector construction: Two Flotillin1 targeting sgRNAs (the amino acid sequence is shown in SEQ ID NO:1 and the nucleotide sequence is shown in SEQ ID NO:2) and two OsImportin α4 targeting sgRNAs (the amino acid sequence is shown in SEQ ID NO:3 and the nucleotide sequence is shown in SEQ ID NO:4) were designed and sequentially ligated into the CRISPR / Cas9 binary vector pYLCRISPR / Cas9Pμbi-H.

[0125] (2) The target vector was transformed into Agrobacterium EHA105 and then transferred into Nipponbare rice using Agrobacterium-mediated transformation. The homozygous Importin α4-Flotillin1 gene knockout mutant was identified targeting the Importin α4 and Flotillin1 genes.

[0126] (3) Molecular identification of Importin α4 mutant rice: DNA was extracted from transgenic plants, primers were designed at both ends of the target sgRNAs, PCR was performed to expand the fragment containing the target region, and sequencing analysis was performed to analyze the targeting effect.

[0127] Example 3. Verification and testing steps of rice mutants

[0128] 1. RNA extraction from rice, cDNA synthesis, and qPCR quantification.

[0129] (1) RNA extraction: RNA was extracted from rice leaves using the Trizol method (Ambion, 15596018). The specific steps are as follows:

[0130] Take 200 mg of rice leaves and place them in a 1.5 mL centrifuge tube. Add 1 mL of Trizol reagent (Ambion, 15596018) and grind the sample thoroughly. Let the sample stand at room temperature for 5 min to allow for complete lysis. Centrifuge at 12000 rpm for 5 min at 4 °C and discard the precipitate. Add 200 μL of chloroform, shake vigorously for 15 s, let stand at room temperature for 15 min, and centrifuge at 12000 rpm for 15 min at 4 °C. The sample will separate into three layers. Transfer the upper aqueous phase to a new 1.5 mL centrifuge tube, add 0.5 mL of isopropanol, gently invert to mix, let stand at room temperature for 5 min, and centrifuge at 12000 rpm for 10 min at 4 °C and discard the supernatant. Add 1 mL of 75% ethanol to the centrifuge tube, shake gently to suspend the precipitate, and centrifuge at 8000 g for 5 min at 4 °C and discard the supernatant. Wash once more with 1 mL of 75% ethanol. Place the centrifuge tube open in a clean bench and air dry at room temperature for 5 min. Add 30 μL of RNase-free... Dissolve the RNA precipitate with ddH2O; take 1 μL and measure the RNA concentration on a Nanodrop 2000. Select samples that are not contaminated by impurities such as proteins, phenols, inorganic salts, and carbohydrates and have a concentration greater than 125 ng / μL for subsequent experiments.

[0131] (2) cDNA synthesis: cDNA was synthesized from RNA using the M-MLV reverse transcription system (Promega, MSA). The specific steps are as follows.

[0132] Add 1 μg of RNA solution to a 0.2 mL PCR tube, bring the volume to 12 μL with double-distilled water (RNase-free), add 1 μL of random primers (random primers are required if cloning or detecting viral genes; Oligo-dT primers can be used if only eukaryotic genes are being cloned), mix thoroughly by pipetting, centrifuge to the bottom of the tube, incubate at 70°C for 60 min, then incubate at 4°C for 10 min; add 5 μL of 5x MLV buffer, 5 μL of dNTPs, 1 μL of reverse transcriptase, and 1 μL of RNase inhibitor to the above tube, mix thoroughly by pipetting, centrifuge to the bottom of the tube, incubate at 42°C for 60 min, then incubate at 75°C for 15 min to terminate the reaction; dilute the reverse transcription product according to experimental requirements and store at -20°C.

[0133] (3) Primer design: The quantitative primer designs for each gene are shown in Table 4.

[0134] Table 4. Primer sequences for the target gene

[0135]

[0136] (4) qPCR quantification: qPCR was performed using Tiangen's Talent qPCR PreMix (FP209-01), and the reaction system is shown in Table 5. A two-step PCR reaction procedure was used, as shown in Table 6. After sealing the tubes, centrifuge to allow the reaction mixture to reach the bottom. Place the reaction system in a Thermo Pikoreal 96 Real-Time PCR System to begin the reaction.

[0137] Table 5. qPCR reaction system

[0138]

[0139]

[0140] Table 6. qPCR reaction procedure

[0141]

[0142] 2. Rice protein extraction

[0143] Proteins were extracted from rice using the RIPA lysis buffer kit (CW2333) from Kangwei Reagents. First, 1g of tissue was added to 5ml of Plant protein extraction reagent (containing 1% protein inhibitor cocktail), and mechanically homogenized for lysis. The mixture was then incubated on ice for 20-30 minutes, followed by centrifugation at 4℃, 13, 400g for 20 minutes. The supernatant was collected and stored at -20℃.

[0144] 3. Western blotting experiment

[0145] Prepare a 10% separating gel and a 5% stacking gel; run at 80V for 30 minutes, and after the sample enters the stacking gel, run at 120V for 1 hour, stopping when the bromophenol blue reaches the green line; prepare 1L of transfer buffer (200ml anhydrous methanol, 11.25g glycine, 3.025g Tris, and ultrapure water to a final volume of 1L); activate the membrane in methanol for about 1 minute; then transfer it to the transfer buffer: pour the transfer buffer into a tray, black side down, and arrange the layers in the following order: black side-sponge-filter paper-gel-membrane-filter paper-sponge-white plate, removing air bubbles with a roller after each layer is added. After assembly, place the membrane in the transfer tank, with the black side facing the black plate and the white plate facing the red plate; place the transfer tank in an ice-water mixture, 100V, 1 hour: seal with 5% skim milk (Bio-Easy, BE6250) for 1 hour, shaking. Bed rotation speed 60 rpm / min: Discard the milk, add 5 ml of fresh milk, add 1.5 μl of primary antibody (1:3000), incubate at room temperature for 2 h or overnight at 4°C, wash 3 times with PBST for 5 min each time; add 5 ml of milk, add 1 μl of secondary antibody (1:5000), incubate at room temperature for 1 h, wash 3 times with PBST for 5-15 min each time; ECL chemiluminescence solution (Thermo, A38556), mix solutions A and B 1:1, add 400 μl to each membrane, and image using a gel imaging system.

[0146] 7. Rice inoculation with RSV experiment

[0147] When rice seedlings reached the 2.5-leaf stage, they were inoculated with the virus. An average of 30 infected planthoppers were inoculated per plant, and the seedlings were encased in microbeetles. Leaves fed on by the infected planthoppers were designated as inoculated leaves, while the remaining rice leaves were designated as system leaves. Two days after inoculation, the infected planthoppers and microbeetles were removed. qPCR was used to quantitatively detect the infection rate on system and inoculated leaves at 1, 4, and 7 days after inoculation, with 8 biological replicates per treatment. Inoculated leaves were collected at 12 hours, and system leaves at 1 day for plant immunofluorescence assays to detect virus spread, with 9-12 biological replicates per treatment. Two infection systems were used to assess rice disease incidence, with an average of 10 and 2 infected planthoppers per plant, encased in microbeetles. Two days after inoculation, the infected planthoppers and microbeetles were removed, and the disease incidence was recorded, with 6 biological replicates per treatment.

[0148] 8. Immunofluorescence experiment on rice leaves

[0149] Rice leaves treated with the virus were fixed overnight at 4°C with 4% paraformaldehyde (Coollab, SL18301); the tissues were transferred into rigid gelatin capsules (Electron Microscopy Sciences, 70102) and embedded in a Japanese Sakura (SAKMRA) medium. 4583) Embedded, air removed, and quick-frozen at -20℃ for 5 min; cut into 10 μm sections using a cryostat (Leica), placed on adhesive slides, and air-dried; incubated in 10% goat serum (Beyotime, CO265) at room temperature for 1 h to block non-specific binding sites; eluted twice with PBST (containing 0.1% Tween 20); diluted the primary antibody (RSVNP mouse monoclonal antibody 1:500) with PBST, incubated overnight at 4℃, eluted three times with PBST, and began light-protected procedures; diluted the secondary antibody (abberior, STORANGE-1002 / 1001) with PBST (1:1000), incubated at room temperature for 3 h, eluted three times with PBST; added 20 μl of 0.25 mg / mL Aniline B1μe to each section. Fluorochrome (Biosμpplies, 100-1) with 100 μl of antifluorescence quencher (BOSTER, AR1109) added, incubated at room temperature for 30 min to stain plasmodesmata; mounted with nail polish, and photographed using a laser confocal microscope (excitation wavelength 390 nm, emission wavelength 480 nm, STORANGE-1002); each treatment had at least 6 biological replicates.

[0150] Example 4. Study on the Flotillin1 mutant in rice

[0151] 1. Transcriptional and protein levels

[0152] The results show that ( Figure 1 At the transcriptional level, the expression level of the Flotillin1 gene was significantly reduced in Flotillin1 mutant rice compared with wild-type rice (WT); at the protein level, the expression level of Flotillin1 protein was significantly reduced in Flotillin1 mutant rice compared with wild-type rice (WT).

[0153] 2. Effects of Flotillin1 gene knockout on rice plant height, thousand-grain weight, and germination rate

[0154] Compared with WT, there were no significant differences in plant height and thousand-grain weight in rice from the Flotillin1 mutant. Figure 2 ).

[0155] 3. Effect of Flotillin1 gene knockout on RSV dispersal rate in rice

[0156] The effect of Flotillin1 knockout on RSV diffusion rate in rice was investigated using plant immunofluorescence technology. The results showed that ( Figure 3 When RSV infected the inoculated leaves for 12 hours, compared with WT, the diffusion rate of RSV in bundle sheath cells, sieve tubes and companion cells in the Flotillin1 mutant was significantly reduced by 55%, 36% and 34%, respectively. When RSV infected the systemic leaves for 1 day, compared with WT, the diffusion rate of RSV in sieve tubes and companion cells in the Flotillin1 mutant was significantly reduced by 64% and 57%, respectively.

[0157] 4. Effect of Flotillin1 gene knockout on RSV NP expression in rice

[0158] The effect of Flotillin1 gene knockout on RSV NP expression in rice was investigated by inoculating rice with highly infected RSV planthoppers. The results showed that ( Figure 4 In the Flotillin1 mutant, compared with WT, there was no significant difference in RSV NP expression 1 day after RSV infection of systemic leaves, but RSV NP expression was significantly reduced by 87% and 53% 4 and 7 days after RSV infection of systemic leaves, respectively.

[0159] 5. Effects of Flotillin1 gene knockout on disease incidence in rice

[0160] The effect of Flotillin1 gene knockout on rice disease incidence was investigated by inoculating rice with highly virulent RSV planthoppers. The results showed that ( Figure 5 In the Flotillin1 mutant, compared with WT, the incidence of RSV was significantly reduced by 20% when the insect population density was 10, and by 30% when the insect population density was 2.

[0161] Example 5. Study of Importin α4 mutant

[0162] 1. Effects of knocking out Importin α4 on rice plant height and thousand-grain weight

[0163] Compared with WT, the Importin α4 mutant showed a significant decrease in both plant height and thousand-grain weight in rice. Figure 6 ).

[0164] 2. Effect of Importin α4 knockout on RSV NP expression in rice

[0165] The effect of knockout of Flotillin1-Importin α4 on the expression level of RSV NPs in rice was investigated by inoculating rice with highly virulent RSV planthoppers. The results showed that ( Figure 7 In the Importin α4 mutant, compared with WT, there was no significant difference in the expression level of RSV NP after 1d and 4d of RSV infection of systemic leaves, but the expression level of RSV NP was significantly reduced by 84% after 7d of RSV infection of systemic leaves.

[0166] 3. Effects of Importin α4 knockout on disease incidence in rice

[0167] The effect of knockout of Importin α4 on rice disease incidence was investigated by inoculating rice with highly virulent RSV planthoppers. The results showed that ( Figure 8 In the Importin α4 mutant, compared with WT, the incidence of RSV was significantly reduced by 24% when the insect population density was 10, and by 27% when the insect population density was 2.

[0168] Example 6. Study of Flotillin1-Importin α4 mutant

[0169] 1. Effects of Flotillin1-Importin α4 knockout on rice plant height and thousand-grain weight

[0170] Compared with WT, there were no significant differences in plant height and thousand-grain weight in rice from the Flotillin1-Importin α4 mutant. Figure 9 ).

[0171] 2. Effect of Flotillin1-Importin α4 knockout on RSV NP expression in rice

[0172] The effect of knockout of Flotillin1-Importin α4 on the expression level of RSV NPs in rice was investigated by inoculating rice with highly virulent RSV planthoppers. The results showed that ( Figure 10 In the Flotillin1-Importin α4 mutant, compared with WT, the expression level of RSV NP was significantly reduced by 82%, 97% and 70% after RSV infection of systemic leaves for 1 day, 4 days and 7 days, respectively.

[0173] 3. Effects of Flotillin1-Importin α4 knockout on disease incidence in rice

[0174] The effect of knockout of Flotillin1-Importin α4 on rice disease incidence was investigated by inoculating rice with highly virulent RSV planthoppers. The results showed that ( Figure 11In the Flotillin1-Importin α4 mutant, compared with WT, the incidence of RSV was significantly reduced by 37% when the insect population density was 10, and by 40% when the insect population density was 2.

[0175] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A method of obtaining rice that is capable of resisting Rice stripe virus, the method comprising: Simultaneously inhibit the transcription or translation of the Flotillin1 gene and the Importin α4 gene in rice, or simultaneously inhibit the expression level of the proteins encoded by the Flotillin1 gene and the Importin α4 gene in rice; wherein, the amino acid sequence of the protein encoded by the Importin α4 gene is shown in SEQ ID NO: 3, and the amino acid sequence of the protein encoded by the Flotillin1 gene is shown in SEQ ID NO:

1.

2. The method as described in claim 1, wherein when several copies of the Flotillin1 gene and the Importin α4 gene are present in the rice, each copy of the Flotillin1 gene and the Importin α4 gene becomes defective.

3. The method of claim 1, wherein the method is implemented by the following steps (a) to (e): (a) Construct a vector containing sgRNA1 and sgRNA2, wherein sgRNA1 can target the Flotillin1 gene and sgRNA2 can target the Importin α4 gene; (b) Transform the vector into Agrobacterium; (c) Infect rice cells with the Agrobacterium; (d) Select rice cells with defective Flotillin1 and Importin α4 genes; (e) Generate rice from the rice cells of step (c) or (d).

4. The method of claim 3, further comprising step (f): after step (e), screening rice varieties that simultaneously possess the defective Flotillin1 gene and Importin α4 gene to obtain rice varieties resistant to rice stripe virus.

5. The method of claim 3, wherein the method has one or more features selected from the following: (1) The sgRNA1 and sgRNA2 are constructed in the same vector or different vectors; (2) The Agrobacterium is EHA105 Agrobacterium.

6. The method of claim 4, wherein in step (f), RNA is extracted from the rice, the RNA is reverse transcribed into cDNA, and the cDNA is amplified using primers to detect nucleotide fragments targeting sgRNA1 and sgRNA2 for screening; or, genomic DNA of the rice is extracted, and the DNA is amplified using primers to detect nucleotide fragments targeting sgRNA1 and sgRNA2 for screening.

7. The method of claim 5, wherein the sgRNA1 and sgRNA2 are constructed in the pYLCRISPR / Cas9Pμbi-H vector.

8. The method of claim 5, wherein the method has one or more features selected from the following: (1) The nucleotide sequence of the sgRNA1 is SEQ ID NO: 5 or SEQ ID NO: 6; (2) The nucleotide sequence of the sgRNA2 is SEQ ID NO: 7 or SEQ ID NO:

8.

9. Use of a molecule that specifically inhibits simultaneously the transcription or the translation of the Flotillin 1 gene and the Importin a4 gene or that specifically inhibits simultaneously the expression level of the proteins encoded by the Flotillin 1 gene and the Importin a4 gene in the manufacture of a kit for obtaining a rice plant capable of resisting the Rice stripe virus, or for increasing the ability of a rice plant to resist the Rice stripe virus; wherein, The amino acid sequence of the protein encoded by the Importin α4 gene is shown in SEQ ID NO: 3, and the amino acid sequence of the protein encoded by the Flotillin1 gene is shown in SEQ ID NO:

1.

10. The use of claim 9, wherein, The molecule in question is a nucleic acid molecule.

11. The use of claim 10, wherein, The nucleic acid molecule is selected from antisense oligonucleotides, dsRNA, siRNA, shRNA, or sgRNA.

12. The use as described in claim 10, wherein, The nucleic acid molecule contains sgRNA1 and sgRNA2, wherein sgRNA1 can target the Flotillin1 gene or a fragment thereof, and sgRNA2 can target the Importin α4 gene or a fragment thereof.

13. The use as described in claim 12, wherein, The nucleotide sequence of the sgRNA1 is SEQ ID NO: 5 or SEQ ID NO:

6.

14. The use as described in claim 12, wherein, The nucleotide sequence of the sgRNA2 is SEQ ID NO: 7 or SEQ ID NO:

8.

15. The use as described in claim 9, wherein, The kit also includes: a vector, Agrobacterium, a culture medium and / or reagents for culturing rice cells or tissues, reagents for extracting rice DNA or RNA, a primer set for amplifying fragments targeting sgRNA1 and sgRNA2 respectively, or any combination thereof.

16. The use as described in claim 15, wherein, The vector is pYLCRISPR / Cas9Pμbi-H.

17. The use as described in claim 15, wherein, The sgRNA1 and sgRNA2 may or may not be contained in the vector.

18. The use as described in claim 15, wherein, The Agrobacterium is EHA105 Agrobacterium.

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