Gene RGA3 for regulating and controlling disease and insect pest resistance and application

Abstract

The invention provides a gene RGA3 for regulating and controlling disease and insect pest resistance and application thereof. The invention discloses a novel gene RGA3 from a gramineous plant. The novel gene RGA3 has the effect of improving the capability of the plant to resist plant diseases and insect pests. The method can be applied to plant variety improvement, and plants with excellent disease and pest resistance are obtained.

Description

Technical Field

[0001] The present invention belongs to the fields of botany and molecular biology, and more particularly, relates to a gene RGA3 for regulating resistance to plant diseases and insect pests and its application. Background Art

[0002] Plants face threats from a variety of herbivorous insects during their growth and development, requiring a complex defense system to combat these attacks. Plants possess constitutive defense systems, including physical barriers such as waxes, cuticles, and lignin on their surfaces. The fundamental building blocks of this defense network are plant genes. Overexpressing or knocking out a gene can affect specific defense pathways, resulting in either enhanced or weakened resistance to insects.

[0003] When a plant's autoimmune system is activated, it initiates an innate immune response, triggering a series of physiological and biochemical reactions and gene expression. This innate immune response includes immune responses (PTI) triggered by pattern recognition receptors on the cell membrane and immune responses (ETI) triggered by plant-specific resistance proteins (R proteins) recognizing receptors and secreting effector proteins. Once the plant's immune response is activated, a complex network of defense signals is woven within the plant, including jasmonic acid (JA), salicylic acid (SA), and H2O2.

[0004] In addition, a variety of other metabolic pathways are also potentially involved in plant resistance to insects, bacteria or fungi.

[0005] At present, the research on the molecular mechanisms of resistance of plants such as grasses to insects, such as planthoppers, is still in its early stages. Discovering genes related to insect resistance and enhancing understanding of the molecular mechanisms of insect resistance will help in the prevention and control of insects in the field. Summary of the Invention

[0006] The purpose of the present invention is to provide a gene RGA3 for regulating resistance to plant diseases and insect pests and its application.

[0007] In a first aspect of the present invention, a method for improving a plant's resistance to pests and diseases (resistance to pests and diseases) or preparing a plant with improved resistance to pests and diseases is provided, comprising: increasing the expression or activity of RGA3 in the plant.

[0008] In one or more embodiments, the pests include insects; preferably, the insects include Hemiptera, Homoptera, Diptera, and Lepidoptera; more preferably, the Hemiptera include Delphacidae, including brown planthopper (Nilaparvata lugens), white-backed planthopper (Sogatella furcifera) and gray planthopper (Lalielphax striatellus).

[0009] In one or more embodiments, the pests and diseases include bacteria, and the bacteria include: Xanthomonas oryzae pv.oryzae, Xoo or similar bacteria thereof; preferably, the similar bacteria include: bacterial leaf spot pathogen (Xanthomonas oryzae pv.Oryzicola, oc), rice bacterial brown spot pathogen (Pseudomonassyringae pv.syringae Van Holl) and rice bacterial base rot pathogen (Erwinia chrysanthemipv.zeae (Sabet) Victria).

[0010] In one or more embodiments, the pests and diseases include fungi, and the fungi include rice blast fungus.

[0011] In one or more embodiments, the RGA3 includes homologs thereof.

[0012] In one or more embodiments, the method comprises: introducing a polynucleotide or construct (such as an expression vector) encoding RGA3 into a plant; or administering an up-regulator of RGA3 to a plant, thereby increasing the expression or activity of RGA3 in the plant.

[0013] In one or more embodiments, the method comprises the steps of: (i) providing Agrobacterium carrying an expression vector containing a polynucleotide encoding RGA3; and (ii) introducing the polynucleotide encoding RGA3 into a plant using Agrobacterium.

[0014] In one or more embodiments, the plant includes: a grass plant; preferably, the grass plant is a food crop; more preferably, it includes: rice, barley, wheat, oats, rye, corn or sorghum.

[0015] In another aspect of the present invention, a use of RGA3 or an up-regulator thereof is provided for improving the ability of plants to resist pests and diseases or preparing plants with improved resistance to pests and diseases.

[0016] In one or more embodiments, the RGA3 comprises:

[0017] (a) a protein having the amino acid sequence shown in SEQ ID NO: 2; or

[0018] (b) a protein derived from (a) formed by substituting, deleting or adding one or more (e.g., 1-20; preferably 1-10; more preferably 1-5) amino acid residues of the amino acid sequence shown in SEQ ID NO: 2, and having the same function as the protein in (a); or

[0019] (c) a protein derived from (a) that has an amino acid sequence that is more than 80% (preferably more than 85%; more preferably more than 90%; more preferably more than 95%; more preferably more than 98% or 99%) identical to the amino acid sequence defined in (a), and that has the same function as the protein in (a); or

[0020] (d) A protein fragment of SEQ ID NO: 2 having the function of the protein in (a).

[0021] In one or more embodiments, the upregulator of RGA3 includes: an expression construct, an expression cassette or an expression vector of RGA3; or a molecule that interacts with RGA3 to promote its expression or activity.

[0022] In another aspect of the present invention, there is provided a use of RGA3 or a polynucleotide encoding the same as a molecular marker for identifying the ability of a plant to resist diseases and insect pests.

[0023] In another aspect of the present invention, a plant cell, tissue or organ is provided, which expresses exogenous RGA3, or an expression cassette containing exogenous RGA3; preferably, the expression cassette comprises: a promoter, a gene encoding RGA3, and a terminator.

[0024] In one or more embodiments, the expression cassette is contained in a construct or expression vector.

[0025] In one or more embodiments, the plant cells, tissues or organs do not directly generate living plants or serve as plant propagation material.

[0026] In another aspect of the present invention, a method for selecting or identifying plants with the ability to resist pests and diseases is provided, the method comprising: identifying the expression or activity of RGA3 in a test plant; if the expression or activity of RGA3 in the test plant is high (e.g., higher than the average value of the plant), the plant is considered to be resistant to pests and diseases.

[0027] In another aspect of the present invention, a method for screening a potential substance for improving a plant's resistance to pests and diseases is provided, the method comprising:

[0028] (1) treating an expression system expressing RGA3 with a candidate substance; and

[0029] (2) detecting the expression or activity of RGA3 in the system; if the candidate substance statistically increases the expression or activity of RGA3, it indicates that the candidate substance is a potential substance for improving (e.g., by 5%, 10%, 15%, 20%, 30% or more, preferably by 50% or more; more preferably by more than 80%) the plant's ability to resist diseases and insect pests.

[0030] In one or more embodiments, the candidate substances include (but are not limited to): regulatory molecules targeting RGA3 protein or its encoding gene, or its upstream or downstream proteins or genes, such as upregulators, interfering molecules (for example, they can interfere with upstream genes that "inhibit RGA3 expression"), agonists, nucleic acid inhibitors, binding molecules, CRISPR constructs, and small molecule compounds.

[0031] In one or more embodiments, the screening method further comprises setting up a control group to clearly distinguish the expression or activity of RGA3.

[0032] In one or more embodiments, the high expression or high activity refers to a statistically significant increase in expression or activity compared to the average value of the expression or activity of the same species or the same plant, such as an increase of 10%, 20%, 40%, 60%, 80%, 90% or more.

[0033] In one or more embodiments, the high expression or high activity refers to a statistically significant increase in expression or activity compared to the average value of the expression or activity of the same species or the same plant, such as an increase of 10%, 20%, 40%, 60%, 80%, 90% or more.

[0034] In one or more embodiments, the high ability to resist pests and diseases means that the ability to resist pests and diseases is statistically significantly higher than that of similar or same plants, such as 10%, 20%, 40%, 60%, 80%, 90% or higher.

[0035] Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 , OsRGA3 overexpression and knockout material detection;

[0037] A: Detection of OsRGA3 gene expression in OsRGA3 overexpressing materials;

[0038] B: Schematic diagram of the osrga3 knockout site.

[0039] Figure 2 , the response of OsRGA3 to brown planthopper feeding and rice blast damage;

[0040] A: Response of OsRGA3 to feeding by brown planthoppers;

[0041] B: Response of OsRGA3 to rice blast.

[0042] Figure 3 , expression of OsRGA3 in different tissues of rice.

[0043] Figure 4 , identification of brown planthopper resistance in OsRGA3 overexpression and knockout materials;

[0044] A: Identification of brown planthopper resistance in single OsRGA3-overexpressing plants;

[0045] B: Identification of brown planthopper resistance in a small population of OsRGA3-OE2;

[0046] C: Mortality statistics of small population of brown planthoppers for resistance identification of OsRGA3-OE2;

[0047] D: Identification of brown planthopper resistance in a small population of OsRGA3-OE10;

[0048] E: Mortality statistics of small population of brown planthopper in resistance identification of OsRGA3-OE10;

[0049] F: Identification of brown planthopper resistance in a small population of OsRGA3-OE11;

[0050] G: Mortality statistics of small populations of brown planthoppers for resistance identification against OsRGA3-OE11;

[0051] H: Identification of resistance of individual plants to brown planthoppers in osrga3;

[0052] I: Identification of brown planthopper resistance in a small population of osrga3;

[0053] J: Mortality statistics for resistance identification of brown planthopper in small populations of osrga3.

[0054] Figure 5 , identification of rice blast resistance in OsRGA3 overexpression and knockout materials;

[0055] A: Photos of lesions on detached leaves of OsRGA3 overexpression and knockout materials 14 days after inoculation with rice blast;

[0056] B: statistics of lesion size;

[0057] C: Detection of rice blast fungus growth.

[0058] Figure 6 , identification of bacterial blight resistance of OsRGA3 overexpression and knockout materials;

[0059] A: Photos of lesions on leaves of OsRGA3 overexpression and knockout materials 14 days after inoculation with bacterial blight;

[0060] B: Statistics of lesion length on leaves of OsRGA3 overexpression and knockout materials 14 days after inoculation with bacterial blight. DETAILED DESCRIPTION

[0061] Based on extensive research and screening, the present invention has revealed a novel plant gene, named RGA3, which has the ability to enhance plant resistance to pests and diseases. This invention can be applied to plant variety improvement to obtain plants with superior pest and disease resistance.

[0062] As used herein, the "plant (crop)" is a plant containing RGA3 or a homolog thereof; preferably, the plant includes, but is not limited to, plants in the grass family, cruciferous plants, solanaceous plants, and euphorbiaceous plants. More preferably, the plant is a grass family. More preferably, the plant is a crop (such as a food crop or cash crop). For example, the "plant" includes, but is not limited to, rice, sorghum, corn, barley, wheat, oats, rye, and the like.

[0063] As used herein, the insects include Hemiptera, Homoptera, Diptera, Lepidoptera (such as the striped stem borer), etc.; preferably, they are planthoppers. The "planthoppers" include but are not limited to: brown planthopper (BPH), white back planthopper (WBPH) and small brown planthopper (SBPH). "Planthoppers" is a family belonging to the order Homoptera. "Planthoppers" have many common characteristics as follows: they are commonly known as planthoppers, all of which are herbivorous, and many species live on grass plants. They are piercing-sucking agricultural pests. They have migratory habits and are the primary pest on rice in my country and many Asian countries. Among them, brown planthoppers are monophagous pests that feed and reproduce on rice and common wild rice. White back planthoppers and gray planthoppers have a wide diet and can harm grass plants such as rice, wheat, and corn.

[0064] As used herein, "exogenous" or "heterologous" refers to the relationship between two or more nucleic acid or protein sequences that are derived from different sources. For example, a promoter is exogenous to a gene of interest if the combination of the promoter and the gene sequence does not normally occur in nature. A particular sequence is "exogenous" to the cell or organism into which it is inserted.

[0065] As used herein, the term "expression cassette" refers to a gene expression system comprising all necessary elements for expressing a polypeptide of interest (e.g., RGA3 in the present invention). It typically includes the following elements: a promoter, a gene sequence encoding the polypeptide, a terminator, and optionally a signal peptide coding sequence. These elements are operably linked.

[0066] The present invention discovered that upregulating RGA3 expression can enhance plant resistance to pests and diseases. In the present invention, the rice-derived RGA3 (OsRGA3) is a polypeptide / gene with LOC_Os11g11770. Sequence variants thereof are also encompassed by the present invention.

[0067] It is understood that, although RGA3 of the present invention is preferably obtained from rice, other polypeptides obtained from other plants that are highly homologous to RGA3 in rice (e.g., having a sequence identity of 60% or more, such as 70%, 75%, 80%, 85%, 90%, 95%, or even 98%) are also within the scope of the present invention, and these polypeptides are also referred to as "homologs" of RGA3. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0068] The present invention also encompasses fragments, derivatives, and analogs of RGA3. As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the same biological function or activity as RGA3 of the present invention. A polypeptide fragment, derivative, or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code; (ii) a polypeptide having a substituent group in one or more amino acid residues; or (iii) a polypeptide formed by fusion of an additional amino acid sequence to a polypeptide sequence (e.g., a leader sequence or secretory sequence, a sequence used to purify the polypeptide, a proprotein sequence, or a fusion protein). These fragments, derivatives, and analogs are well known to those skilled in the art, as defined herein.

[0069] Any biologically active fragment of RGA3 may be used in the present invention. Herein, a biologically active fragment of RGA3 refers to a polypeptide that retains all or part of the functionality of full-length RGA3. Typically, the biologically active fragment retains at least 50% of the activity of full-length RGA3. More preferably, the biologically active fragment retains 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of full-length RGA3.

[0070] In the present invention, the term "RGA3" refers to the polypeptide having the sequence of SEQ ID NO: 2. The term also includes variants of SEQ ID NO: 2 that have the same function as RGA3. These variants include (but are not limited to): deletions, insertions, and / or substitutions of several amino acids (generally 1-50, preferably 1-30, more preferably 1-20, optimally 1-10, and even more preferably 1-8 or 1-5), as well as additions or deletions of one or more amino acids (generally 1-50, preferably 1-30, more preferably 1-20, optimally 1-10, and even more preferably 1-8 or 1-5) at the C-terminus and / or N-terminus (particularly the N-terminus). For example, substitutions with amino acids having similar or similar properties generally do not alter protein function. For another example, additions or deletions of one or more amino acids at the C-terminus and / or N-terminus (particularly the N-terminus) generally do not alter protein function. The term also includes active fragments and active derivatives of RGA3.

[0071] Any protein with high homology to the RGA3 (e.g., 50% or higher homology to the sequence shown in SEQ ID NO: 2; preferably, 60% or higher homology; preferably, 70% or higher homology; preferably, 80% or higher homology; more preferably, 90% or higher homology, such as 95%, 98% or 99% homology) and having the same function as RGA3 is also included in the present invention.

[0072] In the present invention, "RGA3 conservative variant polypeptide" refers to a polypeptide in which at most 20, preferably at most 10, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids with similar or similar properties compared to the amino acid sequence of SEQ ID NO: 2.

[0073] The present invention also relates to a polynucleotide sequence encoding the RGA3 of the present invention or a conservative variant polypeptide 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 shown in SEQ ID NO:1 or a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes a protein having SEQ ID NO:2 but differs from the coding region sequence shown in SEQ ID NO:1.

[0074] The polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: a coding sequence encoding only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0075] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and / or non-coding sequences.

[0076] The present invention also relates to variants of the aforementioned polynucleotides, which encode polypeptides or polypeptide fragments, analogs, and derivatives having the same amino acid sequence as the present invention. These polynucleotide variants may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide, which may contain one or more nucleotide substitutions, deletions, or insertions that do not substantially alter the function of the encoded polypeptide.

[0077] The present invention also relates to a vector comprising the polynucleotide, and a host cell produced by genetic engineering using the vector or the RGA3 coding sequence.

[0078] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. Plant transformation can be carried out using methods such as Agrobacterium transformation or gene gun transformation, for example, the spraying method, leaf disc method, and rice embryo transformation method.

[0079] The present invention also provides a method for improving plants, comprising increasing RGA3 expression in the plant. The improved plant comprises enhancing the plant's ability to resist pests and diseases. Once the function of RGA3 is understood, various methods well known in the art can be used to increase RGA3 expression. For example, an expression unit (such as an expression vector or virus) carrying the RGA3 gene can be delivered to the target site using methods known in the art to induce expression of active RGA3.

[0080] Preferably, a method for preparing a transgenic plant is provided, comprising:

[0081] (1) transferring an exogenous RGA3 encoding polynucleotide into a plant tissue, organ or tissue to obtain a plant tissue, organ or seed transformed with the RGA3 encoding polynucleotide; and

[0082] (2) Regenerating the plant tissue, organ or seed obtained in step (1) into which the exogenous RGA3 encoding polynucleotide has been transferred into a plant plant.

[0083] Other methods for increasing the expression of the RGA3 gene or its homologous genes are well known in the art. For example, the expression of the RGA3 gene or its homologous genes can be enhanced by driving the expression with a strong promoter. Alternatively, the expression of the RGA3 gene can be enhanced by using an enhancer (such as the first intron of the rice waxy gene or the first intron of the Actin gene). Strong promoters suitable for the methods of the present invention include, but are not limited to, the 35s promoter and the Ubi promoter of rice and maize.

[0084] The methods described can be carried out using any suitable conventional means, including reagents, temperatures, pressure conditions, and the like.

[0085] Based on the above new discovery of the inventors, the present invention also provides a use of an up-regulator of RGA3 for improving the ability of plants to resist diseases and insect pests or for preparing plants with improved disease and insect pest resistance.

[0086] As used herein, the RGA3 upregulator includes promoters, agonists, and the like. Any substance that can increase the activity of the RGA3 protein, maintain the stability of the RGA3 protein, promote the expression of the RGA3 protein, promote the secretion of the RGA3 protein, prolong the effective action time of the RGA3 protein, or promote the transcription and translation of RGA3 can be used in the present invention as an effective substance for plant improvement.

[0087] As a preferred embodiment of the present invention, the RGA3 protein upregulator includes (but is not limited to): an expression vector or expression construct capable of expressing (preferably overexpressing) RGA3 upon introduction into cells. Typically, the expression vector comprises a gene cassette containing a gene encoding RGA3 and an expression control sequence operably linked thereto. The terms "operably linked" or "operably linked to" refer to a situation in which a portion of a linear DNA sequence is capable of regulating or controlling the activity of another portion of the same linear DNA sequence. For example, if a promoter controls the transcription of a sequence, it is operably linked to a coding sequence.

[0088] The present invention also relates to the use of RGA3 or its encoding gene as a tracking marker for the progeny of genetically transformed plants. The present invention also relates to the use of RGA3 or its encoding gene as a molecular marker to identify a plant's ability to resist pests and diseases by detecting RGA3 expression in the plant. When evaluating a test plant, the expression level or mRNA level of RGA3 can be measured to determine whether the expression or mRNA level in the test plant is higher than the average for that type of plant. If it is significantly higher, the plant has a higher ability to resist pests and diseases.

[0089] Therefore, the present invention provides a method for specifically identifying the ability of a plant to resist pests and diseases, comprising: identifying the expression of RGA3 in the test plant; if the test plant has high expression of RGA3, it is a plant with the ability to resist pests and diseases.

[0090] Those skilled in the art may employ any of a variety of techniques known in the art or currently under development to perform nucleic acid sequence analysis or protein analysis, all of which are encompassed by the present invention. Examples of such methods include, but are not limited to, sequencing, PCR amplification, probe analysis, hybridization, restriction enzyme analysis, immunohistochemistry, and the like.

[0091] Being able to identify a plant's ability to resist diseases and pests in the early stages of planting can greatly facilitate plant breeding.

[0092] Knowing the function and molecular mechanism of RGA3 can be used to conduct targeted screening of plants. This new discovery can also be used to screen for potential substances that regulate RGA3 and thus regulate plant resistance to pests and diseases.

[0093] The present invention provides a method for screening a potential substance for improving the ability of plants to resist diseases and insect pests, the method comprising: (1) treating an expression system with a candidate substance, wherein the system expresses RGA3; and (2) detecting the expression or activity of RGA3 in the system; if the candidate substance statistically improves the expression or activity of RGA3, it indicates that the candidate substance is a potential substance for improving the ability of plants to resist diseases and insect pests.

[0094] Methods for screening for substances that act on proteins or genes, or specific regions thereof, as targets are well known in the art and can be used in the present invention. The candidate substances can be selected from the group consisting of peptides, polymeric peptides, peptidomimetics, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, and nucleic acid sequences. Depending on the type of substance to be screened, those skilled in the art will appreciate how to select an appropriate screening method.

[0095] After large-scale screening, a class of potential substances that specifically act on RGA3 or genes related to its upstream and downstream pathways can be obtained.

[0096] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Science Press, or according to the conditions recommended by the manufacturer.

[0097] Materials and methods

[0098] 1. Plant materials

[0099] The rice used in this study was the japonica rice variety (Oryza sativa L. subsp. Japonica) Zhonghua 11. The rice was grown in Shanghai fields in the summer using standard rice field cultivation and management practices. In the winter, the rice was cultivated in an artificial climate chamber at the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences. The temperature was 29°C ± 1°C, the humidity was 50-70% ± 5%, and the photoperiod was 7:00 AM to 7:00 PM.

[0100] 2. OsRGA3 sequence information

[0101] OsRGA3 nucleotide sequence (coding sequence) (SEQ ID NO: 1):

[0102]

[0103] OsRGA3 amino acid sequence (SEQ ID NO: 2):

[0104]

[0105] 3. Construction of overexpression plants (OsRGA3-OE)

[0106] An overexpression recombinant vector was constructed using the p1301-35S-Nos vector via homologous recombination. The OsRGA3 coding sequence was inserted behind the 35S promoter to form an expression cassette. This expression cassette was then transformed into the wild-type rice variety Zhonghua 11 using Agrobacterium-mediated genetic transformation to obtain the OsRGA3 overexpression material, OsRGA3-OE.

[0107] 4. Construction of OsRGA3-CAS9 vector

[0108] Using the primers designed in Table 1, the primers were first annealed to form double-stranded fragments, which were then constructed into a pOs-sgRNA vector and then transferred to a pH-Ubi-cas9 vector. For detailed information about the vector, refer to the literature (Miao, J. et al. (2013); Targeted mutagenesis in rice using CRISPR-Cas system. Cell Res 23, 1233-1236).

[0109] Table 1. OsRGA3 gene-specific sgRNA sequences (underlined bases are linkers)

[0110]

[0111] 5. Rice genetic transformation

[0112] The main method was carried out according to the literature (Hiei, Y. et al. (1994); Efficient Transformation of Rice (Oryza-Sativa L) Mediated by Agrobacterium and Sequence-Analysis of the Boundaries of the T-DNA. Plant Journal 6, 271-282).

[0113] 6. Identification method of rice plant resistance to brown planthopper

[0114] After germination, rice seeds are sown individually in small plastic pots and grown normally. After about one month, when the seedlings are about to enter the tillering stage, they are tested for insect resistance. Prepare a permeable plastic cover 40 cm high and approximately 8 cm in diameter, with a 6 x 10 cm ventilation opening on one side. Cover it with gauze. Cover the seedlings with the cover and the gauze on top. For testing, place approximately 15 third-instar nymphs (native brown planthopper larvae) in each cover.

[0115] Count the worms once the next day.

[0116] Under normal growth management conditions, wait 5-8 days and observe the survival of the seedlings.

[0117] 7. Identification of Brown Planthopper Resistance in Small Rice Populations

[0118] After germination, rice seeds were planted in blue plastic boxes, with 10 plants per row, for a total of five rows. The control and experimental groups were planted symmetrically. When the rice reached the two-leaf, one-heart stage, they were inoculated with 10 brown planthoppers per plant.

[0119] After access, the growth of rice plants was observed every day, photos were taken and statistics were collected, and three controls were set up.

[0120] 8. Identification method of rice blast resistance

[0121] Rice blast fungus (Magnporthe oryzae) TH12 was used for inoculation, and spores were grown on complete agarose medium at 28°C with 12 h light and 12 h dark conditions for 10 days.

[0122] Before inoculation, spores were collected with sterile water and the spore concentration was adjusted to 1×10 5 spores / ml.

[0123] Select rice seedlings at the four-leaf stage and cut 6-cm-long leaves from the same part of the seedling. Place the leaves in a petri dish and cover both ends with moistened cotton. Use a sterile pipette tip to gently poke the leaves at 1.5-cm intervals, creating three epidermal wounds per leaf. Apply 5 μl of the prepared spore suspension to the wounds.

[0124] Place the cells in a 25°C constant temperature and light incubator, treat them in the dark for 24 hours, and then culture them under 14 hours of light and 10 hours of darkness for 14 days. Observe the disease occurrence and take photos to count the results.

[0125] 9. Identification method of rice resistance to bacterial blight

[0126] Xanthomonas oryzae pv.oryzae, Xoo) PXO99A was cultured on PSA medium at 28°C for 2-3 days, and the bacterial solution was diluted with sterile water to OD 600 =0.5.

[0127] Cut and inoculate rice leaves 1-2 cm away from the tip. The number of leaves cut after the scissors are soaked in the bacterial solution each time should not exceed 3. Inoculate 20 leaves for each rice material. Observe and count the length of the lesions after 14 days.

[0128] Example 1: Construction of OsRGA3 overexpression and knockout materials

[0129] The sequence number of the OsRGA3 gene is LOC_Os11g11770 (http: / / rice.plantbiology.msu.edu / ).

[0130] The inventors constructed the p1301-35S-Nos vector by homologous recombination and transformed it into the wild-type rice variety Zhonghua 11 using Agrobacterium-mediated genetic transformation to obtain OsRGA3 overexpression materials OsRGA3-OE2, OsRGA3-OE10 and OsRGA3-OE11.

[0131] Gene expression analysis showed that OsRGA3 was upregulated by ten to several dozen times in these three strains ( Figure 1 A).

[0132] The inventors also used CRISPR / Cas9 technology to edit the OsRGA3 gene. They first designed an OsRGA3-specific sgRNA and, based on the sgRNA sequence, designed specific primers (Table 1). After annealing, the double-stranded construct was ligated into a Crisper (UBI, OsU6) vector. The transgenic plant was then transformed into the wild-type rice variety Zhonghua 11 via Agrobacterium-mediated genetic transformation. Molecular biological analysis revealed several homozygous mutant plants.

[0133] The inventors analyzed one of the homozygous strains, osrga3. The sequence information of osrga3 is as follows: Figure 1 As shown in B, compared with the wild type, this strain has an "A" inserted at one site in the genome, which results in a frameshift mutation.

[0134] Example 2: OsRGA3 expression response to brown planthopper feeding and rice blast infestation

[0135] To detect whether the OsRGA3 gene plays a role in rice disease and pest resistance, the inventors detected the expression level of OsRGA3 in wild-type rice ZH11 after being attacked by brown planthoppers and rice blast (TH12).

[0136] The response of OsRGA3 to brown planthopper (BPH) feeding is shown in Figure 2 A. The results showed that the expression level of OsRGA3 was upregulated after feeding by brown planthoppers, indicating that OsRGA3 responded to feeding by brown planthoppers.

[0137] The response of OsRGA3 to rice blast (TH12) damage is as follows Figure 2 B.

[0138] The results showed that the expression level of OsRGA3 was upregulated after rice blast infection, indicating that OsRGA3 responded to rice blast infection.

[0139] Example 3: Expression of OsRGA3 in Different Tissues of Rice

[0140] To further investigate whether OsRGA3 may play a role in disease and pest resistance, the inventors detected the expression of this gene in different tissues of rice, including roots, stems, leaves, leaf sheaths, and spikes.

[0141] The results showed that OsRGA3 was expressed in all these tissues, with the highest expression in stems, sheaths and leaves ( Figure 3 ).

[0142] The stems and leaf sheaths are the main feeding parts of brown planthoppers, and the leaves are the main parts of rice damaged by rice blast and bacterial blight, which further suggests that OsRGA3 is involved in the process of rice disease and pest resistance.

[0143] Example 4: OsRGA3 gene positively regulates rice resistance to brown planthopper

[0144] The inventors performed phenotypic identification of OsRGA3-OE2, OsRGA3-OE10, OsRGA3-OE11, and osrga3 plants using single plant and small group brown planthopper resistance identification methods.

[0145] The results showed that plants overexpressing OsRGA3 showed obvious resistance to brown planthoppers in both single plant and small population identifications ( Figure 4 AG).

[0146] The results also showed that plants with homozygous mutations in osrga3 showed a sensitive phenotype to brown planthoppers ( Figure 4 HJ).

[0147] Therefore, OsRGA3 positively regulates rice resistance to brown planthopper.

[0148] Example 5: Rice leaves overexpressing OsRGA3 exhibit blast resistance

[0149] The inventors used scratch inoculation of detached rice leaves to identify the resistance of rice plants overexpressing and knockout OsRGA3 to the rice blast strain TH12. The results showed that 14 days after inoculation with rice blast, the lesion size of rice leaves overexpressing OsRGA3 was significantly smaller than that of wild-type rice, while there was no significant difference between rice leaves with knockout OsRGA3 and wild-type rice ( Figure 5 A and B).

[0150] The biomass of rice blast fungi on rice leaves after inoculation with rice blast was measured. The results showed that 14 days after inoculation with rice blast fungi, the biomass of rice blast fungi in leaves of overexpressing OsRGA3 was significantly lower than that of wild type, while there was no significant difference between the knockout material and the wild type ( Figure 5 C).

[0151] In summary, the leaves of rice plants overexpressing OsRGA3 showed a blast-resistant phenotype.

[0152] Example 6: OsRGA3 knockout rice leaves are sensitive to bacterial blight pathogen

[0153] The inventors inoculated rice leaves with bacterial blight pathogens using the leaf clipping method, measured and counted the leaf lesion lengths 14 days later, and identified the bacterial blight resistance phenotypes of OsRGA3 overexpression and knockout materials.

[0154] The results showed that 14 days after inoculation with bacterial blight, the lesion length of rice leaves with OsRGA3 knockout was significantly longer than that of the wild type, while there was no significant difference between the leaves of rice with OsRGA3 overexpression and the wild type ( Figure 6 A and B).

[0155] In summary, OsRGA3 knockout rice leaves are sensitive to bacterial blight pathogen.

[0156] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the scope of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be based on the appended claims. At the same time, all documents mentioned in this application are cited as references in this application, just as if each document was cited as a reference individually.

Claims

1. A method for improving a plant's resistance to pests and diseases or preparing a plant with improved resistance to pests and diseases, comprising: Increase the expression or activity of RGA3 in plants.

2. The method according to claim 1, wherein The pests include insects; preferably, the insects include Hemiptera, Homoptera, Diptera, and Lepidoptera; more preferably, the Hemiptera include Delphacidae, including brown planthopper (Nilaparvata lugens), white-backed planthopper (Sogatella furcifera) and gray planthopper (Lalielphax striatellus); or The pests and diseases include bacteria, including: Xanthomonas oryzaepv.oryzae (Xoo) or its similar bacteria; preferably, the similar bacteria include: bacterial leaf spot pathogen (Xanthomonas oryzaepv.Oryzicola, oc), rice bacterial brown spot pathogen (Pseudomonas syringae pv.syringae VanHoll) and rice bacterial basal rot pathogen (Erwinia chrysanthemipv.zeae (Sabet) Victria); or The pests and diseases include fungi, and the fungi include rice blast fungi.

3. The method according to claim 1, wherein The method comprises: introducing a polynucleotide or construct encoding RGA3 into a plant; or administering an up-regulator of RGA3 to the plant, thereby increasing the expression or activity of RGA3 in the plant.

4. The method according to claim 1, wherein The method comprises the steps of: (i) providing Agrobacterium carrying an expression vector containing a polynucleotide encoding RGA3; (ii) using Agrobacterium to introduce the polynucleotide encoding RGA3 into plants.

5. The method according to claim 1, wherein The plants include: grass plants; preferably, the grass plants are food crops; more preferably include: rice, barley, wheat, oats, rye, corn or sorghum.

6. Use of RGA3 or an upregulator thereof for improving the ability of plants to resist pests and diseases or preparing plants with improved resistance to pests and diseases.

7. The use according to claim 6, characterized in that The RGA3 includes: (a) a protein having the amino acid sequence shown in SEQ ID NO: 2; or (b) a protein derived from (a) formed by substituting, deleting or adding one or more amino acid residues in the amino acid sequence of SEQ ID NO: 2 and having the same function as the protein in (a); or (c) a protein derived from (a) that has an amino acid sequence that is more than 80% identical to the amino acid sequence defined in (a) and has the same function as the protein in (a); or (d) A protein fragment of SEQ ID NO: 2 having the function of the protein in (a).

8. The use according to claim 6, characterized in that The up-regulator of RGA3 includes: an expression construct, an expression cassette or an expression vector of RGA3; and a molecule that interacts with RGA3 to promote its expression or activity.

9. Use of RGA3 or a polynucleotide encoding the same as a molecular marker for identifying the ability of a plant to resist diseases and insect pests.

10. A plant cell, tissue or organ expressing exogenous RGA3, or an expression cassette comprising exogenous RGA3; preferably, the expression cassette comprises: Promoter, RGA3 encoding gene, terminator; preferably, the expression cassette is contained in a construct or expression vector. In one or more embodiments, the plant cells, tissues or organs do not directly generate living plants or serve as plant propagation material.

11. A method for selecting or identifying plants having the ability to resist pests and diseases, the method comprising: The expression or activity of RGA3 in the test plant is identified. If the expression or activity of RGA3 in the test plant is high, the test plant is a plant with the ability to resist diseases and insect pests.

12. A method for screening a potential substance for improving a plant's resistance to pests and diseases, the method comprising: (1) treating an expression system expressing RGA3 with a candidate substance; and (2) detecting the expression or activity of RGA3 in the system; if the candidate substance statistically increases the expression or activity of RGA3, it indicates that the candidate substance is a potential substance for improving the ability of plants to resist diseases and insect pests; Preferably, the candidate substances include: regulatory molecules, agonists, nucleic acid inhibitors, binding molecules, CRISPR constructs, and small molecule compounds targeting RGA3 protein or its encoding gene, or its upstream or downstream proteins or genes.

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