Use of spd6 gene as a target for regulating disease resistance in plants

Abstract

This invention provides an application of the SPD6 gene as a regulatory target for plant disease resistance. In gramineous plants, downregulators targeting SPD6 enhance disease resistance. This invention offers a new approach for plant breeding and has promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of plant stress resistance; more specifically, this invention relates to the application of the SPD6 gene as a regulatory target for plant disease resistance. Background Technology

[0002] Rice (Oryza sativa) is one of the world's most important crops, providing food and nutrition for more than half of the global population. However, rice production faces multiple threats. Besides abiotic stresses such as drought, floods, and salinity, it is also threatened by diseases caused by pathogenic microorganisms such as bacteria and fungi. Plant diseases can cause significant yield reductions or even crop failure, seriously affecting global food security. Among these, rice blast, caused by the semi-living parasitic fungus Magnaporthe oryzae (M. oryzae), and bacterial leaf blight, caused by the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo), are major rice diseases that seriously threaten rice yield and quality. Rice blast is widely distributed in rice-cultivating countries and regions and is one of the most important diseases affecting rice. In recent years, its affected area has been expanding annually, and its severity has been increasing, becoming one of the main obstacles to high and stable rice yields.

[0003] Currently, traditional chemical control and the planting of resistant varieties are commonly used in production to control diseases. While the large-scale and widespread use of pesticides has mitigated the impact of pests and diseases to some extent, it has also brought new challenges to the ecological environment and food safety. Therefore, cultivating new disease-resistant varieties is of great significance for improving rice yield and quality. Identifying and isolating pathogenic genes and elucidating the disease resistance regulatory mechanisms are the molecular basis for cultivating disease-resistant varieties. However, due to the monoculture of rice and the complexity and highly variable pathogenicity of the physiological races of rice blast fungus, newly bred superior varieties generally lose their resistance after 3-5 years of large-scale promotion. Therefore, finding and utilizing resistance genes with broad-spectrum resistance is currently the most effective and economical measure for controlling rice blast and bacterial blight.

[0004] Traditional disease-resistant breeding relies on resistance identification and plant phenotypic selection, requiring extensive experience and years or even decades of time. Conventional breeding methods often struggle to select varieties possessing multiple disease-resistant genes or exhibiting durable resistance to various diseases. Therefore, identifying genes resistant to multiple diseases and improving rice's resistance to a wide range of diseases is a significant current need in agricultural production. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the SPD6 gene as a target for regulating plant disease resistance.

[0006] In a first aspect of the invention, a method for improving the disease resistance of grass plants is provided, the method comprising downregulating SPD6 in grass plants.

[0007] In another preferred embodiment, the method for improving the disease resistance of grass plants is a transgenic method.

[0008] In a preferred embodiment, downregulating SPD6 in grasses includes downregulating its expression or activity.

[0009] In another preferred embodiment, the downsizing indicates a significant downsizing, such as a downsizing of 20%, 40%, 60%, 80%, 90%, or more.

[0010] In another preferred embodiment, the downregulation of SPD6 includes: knocking out or silencing the SPD6 gene in plants, or inhibiting the activity of the SPD6 protein; preferably, it includes (but is not limited to): knocking out the SPD6 gene using gene editing methods (such as gene editing based on the CRISPR system), silencing SPD6 using interfering molecules that specifically interfere with SPD6 gene expression, knocking out the SPD6 gene using homologous recombination, or inhibiting SPD6 expression by ultraviolet stress; preferably, knocking out the SPD6 gene using gene editing methods, targeting its third exon; more preferably, causing a 1 bp insertion mutation between the 52 bp and 53 bp sites of the third exon or a four-base deletion mutation between the 53 bp and 56 bp sites of the third exon; more preferably, gene editing is performed using sgRNA with the nucleotide sequence shown in SEQ ID NO:4.

[0011] In another preferred embodiment, the interfering molecule is a dsRNA, antisense nucleic acid, small interfering RNA, microRNA, or VIGS molecule that targets the SPD6 coding gene or its transcript for suppression or silencing, or a construct that can express or form the dsRNA, antisense nucleic acid, small interfering RNA, microRNA, or VIGS molecule.

[0012] In another preferred embodiment, the down-adjusting agent is introduced into plants using Agrobacterium-mediated transformation; preferably, the method includes: (1) providing Agrobacterium carrying the down-adjusting agent; (2) contacting plant cells, tissues or organs with the Agrobacterium in step (1) to transfer the down-adjusting agent into the plant; and (3) selecting plants that have been transferred with the down-adjusting agent.

[0013] In another preferred embodiment, the disease resistance is the plant's ability to resist pathogens, including fungi or bacteria.

[0014] In another preferred embodiment, the fungus includes: rice blast fungus.

[0015] In another preferred embodiment, the bacteria include Xanthomonas (bacterium fulvicii).

[0016] In another preferred embodiment, the SPD6 is selected from: (a) a protein with the amino acid sequence of SEQ ID NO:3; (b) a protein derived from (a) having the function of the protein in (a) formed by substituting, deleting, or adding one or more (e.g., 1-20; preferably 1-15; more preferably 1-10, such as 5, 3) amino acid residues of the amino acid sequence of SEQ ID NO:3; or (c) a protein derived from (a) having the function of the protein in (a) having more than 80% (preferably more than 85%; more preferably more than 90%; more preferably more than 95%, such as 98%, 99%) homology to the protein sequence defined in (a); or (d) a protein formed by adding a tag sequence to the N or C terminus of the protein in (a), (b), or (c), or by adding a signal peptide sequence to the N terminus of the protein.

[0017] In another preferred embodiment, the SPD6 comprises a protein encoded by SEQ ID NO:1 or SEQ ID NO:2.

[0018] In another preferred embodiment, the SPD6 includes its homologs.

[0019] In another preferred embodiment, the invention also includes a polynucleotide encoding the preceding SPD6.

[0020] In another aspect of the invention, an application of SPD6 is provided, for use as a downregulation target to enhance the disease resistance of grass plants; or for screening reagents that target SPD6 to enhance the disease resistance of grass plants.

[0021] In another aspect of the invention, the use of an SPD6 down-adjusting agent is provided for improving the disease resistance of gramineous plants; preferably, the SPD6 down-adjusting agent includes (but is not limited to): a down-adjusting agent that knocks out or silences the SPD6 gene or inhibits the activity of the SPD6 protein.

[0022] In a preferred embodiment, the interfering molecule is a dsRNA, antisense nucleic acid, small interfering RNA, microRNA, or VIGS molecule that targets the encoding gene of SPD6 or its transcript for suppression or silencing, or a construct that can express or form the dsRNA, antisense nucleic acid, small interfering RNA, microRNA, or VIGS molecule.

[0023] In another preferred embodiment, the SPD6 down-adjustment agent includes (but is not limited to): a gene editing reagent for knocking out the SPD6 gene (such as a gene editing reagent based on the CRISPR system), an interfering molecule that specifically interferes with the expression of the SPD6 gene, and a reagent for knocking out the SPD6 gene based on homologous recombination; more preferably, the SPD6 down-adjustment agent includes a gene editing reagent targeting the third exon of the SPD6 gene; more preferably, the gene editing reagent is sgRNA with the nucleotide sequence shown in SEQ ID NO:4.

[0024] In another preferred embodiment, the disease resistance is the plant's ability to resist pathogens, including fungi or bacteria; preferably, the fungi include: rice blast fungus; preferably, the bacteria include: bacterial blight fungus.

[0025] In another preferred embodiment, the grass family includes cereal plants, or the SPD6 is derived from cereal plants; preferably, the grass family includes (but is not limited to): rice, wheat, millet, foxtail millet, corn, sorghum, millet, barley, rye, oats, and short-stalked grass.

[0026] In another aspect of the present invention, a method for screening reagents to enhance the disease resistance of gramineous plants is provided, the method comprising:

[0027] (1) Add the analyte to a system containing SPD6;

[0028] (2) Observe the expression or activity of SPD6 in the system of (1); if the analyte inhibits (preferably statistically inhibits; such as reducing by more than 20%, more preferably by more than 50%, and even more preferably by more than 80%) the expression or activity of SPD6, it indicates that the analyte is a reagent to improve the disease resistance of grass plants.

[0029] In a preferred embodiment, the method further includes setting up a control group and a test group to observe the difference between the test group and the control group for the analyte.

[0030] In another preferred embodiment, the analyte includes (but is not limited to): interfering molecules designed for SPD6, or its upstream or downstream proteins or genes, nucleic acid inhibitors, binding molecules (such as antibodies or ligands), small molecule compounds (such as hormones), etc.

[0031] In another preferred embodiment, the system is selected from: cell system (cell culture system), subcellular system, solution system, plant tissue system, and plant organ system.

[0032] In another preferred embodiment, the method further includes conducting further cell experiments and / or transgenic experiments on the obtained potential substances to further identify substances from the test material that are highly effective in improving plant disease resistance.

[0033] In another aspect of the invention, a use of SPD6 is provided for use as a molecular marker to identify disease resistance in grasses.

[0034] In another aspect of the present invention, a method for specifically selecting plants with enhanced disease resistance is provided, the method comprising: identifying the expression of SPD6 in a test plant, wherein if the SPD6 expression of the test plant is significantly lower than the average SPD6 expression value of the same type (or species) of plant, then it is (potentially) a plant with enhanced disease resistance.

[0035] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0036] Figure 1 SPD6-KO produces an autoimmune phenotype.

[0037] (A) Vectors were constructed using the CRSPR / CAS9 system to obtain SPD6 knockout lines under the Nipponbare (NIP) background. (B) qRT-PCR was used to detect the expression of SPD6 in NIP, SPD6-OE#1, SPD6-OE#2, SPD6-KO#1, and SPD6-KO#2 rice materials. OsActin1 was used as an internal control for analysis, and the values ​​are expressed as mean ± standard deviation (n = 3). (C) SPD6-KO showed a lesion-like phenotype. (D) DAB staining of NIP, SPD6-OE, and SPD6-KO materials showed the accumulation of H2O2 in the brown polymer reaction. The accumulation of H2O2 in SPD6 leaves was significantly higher than that in SPD6-OE and SPD6-KO.

[0038] Figure 2 SPD6 itself can generate interactions.

[0039] (A) Transient expression of SPD6-GFP transgenic material in root tips and tobacco plants. Confocal microscopy revealed that SPD6 protein was located in the cell membrane and nucleus. (B) Luciferase complementation assay verified that SPD6 itself generates interactions.

[0040] Figure 3 SPD6 negatively modulates the resistance of rice to bacterial blight.

[0041] (A)(B) Phenotypic and lesion length statistics of NIP, SPD6-OE, and SPD6-KO materials after inoculation with PXO99A and J18 races, respectively, 14 days later. Scale bar, 2cm. Student's t-test, (**, P<0.01; ***, P<0.005; ****, P<0.0001).

[0042] Figure 4 SPD6 negatively regulates rice blast resistance.

[0043] (A) Phenotypic analysis of NIP, SPD6-OE, and SPD6-KO materials after spray inoculation with rice blast race TH12 for 5 days. Scale bar, 1 cm. The growth of rice blast fungus was calculated using qRT-PCR with rice Ubiquitin as an internal control against the inoculated leaf fungus MoPOT2. (B)(C)(D) Phenotypic analysis of NIP, SPD6-OE, and SPD6-KO materials after in vitro inoculation with TH12, GUY11, and YN2 for 5 days, respectively. Scale bar, 1 cm. The length of lesions was measured, and the growth of rice blast fungus was calculated using qRT-PCR. Statistical analysis was performed using Student's test (*, P<0.05; **, P<0.01; ***, P<0.005; ****, P<0.0001). Detailed Implementation

[0044] Through in-depth research and screening, the inventors have revealed a novel target, SPD6, for regulating disease resistance in gramineous plants. Within the plant, inhibitors targeting SPD6 enhance disease resistance. This invention provides a new approach for plant breeding and has promising application prospects.

[0045] As used herein, "plant" includes plants that express SPD6 protein or its homologous protein, or plants whose genome contains the SPD6 gene or its homologous gene. Based on knowledge in the art, plants expressing SPD6 or its homologous protein possess the mechanism of action claimed in this invention and can achieve the technical effects claimed in this invention. The plant can be a monocotyledonous or dicotyledonous plant. In some preferred embodiments, the plant is a crop, preferably a "cereal crop," which is a crop with grains (ears). In some preferred embodiments, the "cereal crop" can be a grass (Poaceae); preferably, the grass includes, but is not limited to, rice, wheat, millet, foxtail millet, corn, sorghum, foxtail millet, barley, rye, oats, and *Brachys edulis*.

[0046] As used herein, “homology” includes homologous proteins or genes of SPD6 in multiple species.

[0047] As used in this article, "exogenous" or "heterogeneous" refers to the relationship between two or more nucleic acid or protein sequences from different sources. For example, if the combination of a promoter and a target gene sequence is not naturally occurring, then the promoter is exogenous to the target gene. A particular sequence is "exogenous" to the cell or organism into which it is inserted.

[0048] As used herein, the terms “reduction,” “decrease,” “downregulation,” “reduction,” “inhibition,” “weakening,” etc., are interchangeable and, in their application, should mean a reduction of at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 50%, 80%, 100%, or more significantly, compared to a control plant, control gene, or control protein as defined herein.

[0049] As used herein, the terms “enhance,” “increase,” “upregulate,” “enlarge,” “promote,” “strengthen,” etc., are interchangeable and, in their application, should mean an increase of at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 50%, 80%, 100%, or more significant, compared to a control plant, control gene, or control protein as defined herein.

[0050] As used in this article, the terms "improved disease resistance", "enhanced antibacterial ability" or "enhanced antibacterial ability" refer to the plant's resistance to Bacillus subtilis.

[0051] Regarding "control plants," selecting appropriate control plants is a routine part of experimental design. These can include corresponding wild-type plants or transgenic plants without the target gene. Control plants are generally the same plant species or even varieties of the same species or class as the plant being evaluated. Control plants can also be individuals from transgenic plants that have lost their transgenic components due to segregation. As used in this article, control plants refer not only to whole plants but also to plant parts, including seeds and seed portions.

[0052] As used in this invention, overexpression, high expression, or high activity refers to a statistically significant increase in expression or activity compared to the average expression or activity of similar or identical plants, such as an increase of 10%, 20%, 40%, 60%, 80%, 90%, or higher.

[0053] The inventors discovered a dwarf rice mutant exhibiting a spontaneous cell death phenotype during their research. Sequencing analysis revealed that this mutant was caused by a mutation in the SPD6 gene. Inoculation with rice blast and bacterial blight showed that the mutant exhibited high resistance to both diseases. Therefore, SPD6 plays a crucial role in rice disease resistance, making it a potential new breeding target.

[0054] In gramineous plants such as rice, SPD6 is located on chromosome 6 and regulates spikelet dwarfing during growth and development. SPD6 encodes a leucine carboxymethyltransferase. Its role in immune regulation has not been disclosed in the art. This invention proposes that SPD6 negatively regulates disease resistance in rice, making it an important target for breeding applications.

[0055] In this invention, the SPD6 protein can be a protein with an amino acid sequence like SEQ ID NO:3. This invention also includes variants having the same function as the SPD6 protein. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and additions or deletions of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, adding one or more amino acids at the C-terminus and / or N-terminus generally does not alter the function of the protein. This invention also provides analogs of the protein. These analogs differ from the natural protein in that they may differ in amino acid sequence, in modifications that do not affect the sequence, or both. These proteins include natural or induced genetic variants. Induced variants can be obtained through various techniques, such as random mutagenesis via radiation or exposure to a mutagen, site-directed mutagenesis, or other known molecular biology techniques. It should be understood that the proteins of this invention are not limited to the representative proteins exemplified above. The polynucleotide encoding SPD6 can be in DNA or RNA form. Polynucleotides encoding the mature SPD6 protein include: a coding sequence encoding only the mature protein; a coding sequence of the mature protein and various additional coding sequences; a coding sequence of the mature protein (and optional additional coding sequences); and non-coding sequences.

[0056] Variations of the SPD6 protein also include (but are not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 1-100 or 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8 or 1-5), and additions or deletions of one or more amino acids (typically up to 20, preferably up to 10, and more preferably up to 5) at the C-terminus and / or N-terminus. Any protein with high homology to the SPD6 protein (e.g., 50% or higher, 60% or higher, 70% or higher homology to the protein sequence shown in SEQ ID NO:3; preferably 80% or higher homology; more preferably 90% or higher homology, such as 95%, 98%, or 99% homology) and having the same function as the SPD6 protein is also included in this invention. Proteins derived from species other than rice that share high homology with the sequence shown in SEQ ID NO:3, or that play the same or similar roles in the same or similar regulatory pathways, are also included in this invention.

[0057] The polynucleotide (gene) of SPD6 can be a natural gene from a plant or a degenerate sequence thereof. This invention also relates to vectors containing the said polynucleotide, and host cells genetically engineered using said vectors.

[0058] In a specific embodiment of the present invention, the field inoculation method for rice bacterial blight, as well as the spray inoculation and in vitro inoculation methods for rice blast, reported in existing literature, were used to inoculate transgenic lines of SPD6 knockout materials and overexpression materials, and their disease resistance was observed. The participation of SPD6 in rice in resistance to the bacterial disease rice bacterial blight and the fungal disease rice blast was identified.

[0059] In specific embodiments of the present invention, it was demonstrated that SPD6 knockout lines in rice exhibited resistance to bacterial blight after inoculation with bacterial blight races PXO99A and J18. Simultaneously, SPD6 knockout lines also showed resistance to rice blast races TH12, GUY11, and YN2 after inoculation, indicating that SPD6 plays a crucial role in regulating rice disease resistance.

[0060] Based on the inventor's discovery, a method for improving plant disease resistance is provided, comprising: downregulating SPD6 in plants.

[0061] According to the explanation of the present invention, after learning about the regulatory mechanism of SPD6 on plant disease resistance, various methods well known to those skilled in the art can be used to reduce or eliminate the expression of SPD6, such as delivering an expression unit (e.g., an expression vector or virus) carrying the antisense SPD6 gene to the target site, so that cells or plant tissues do not express or reduce the expression of SPD6 protein; or knocking out the SPD6 gene.

[0062] As one embodiment of the present invention, the expression of the SPD6 gene in plants can be downregulated by knocking out the SPD6 gene.

[0063] In one embodiment of the present invention, the CRISPR / Cas9 system can be used for gene editing to knock out the SPD6 gene, thereby improving plant disease resistance. Suitable sgRNA target sites lead to higher gene editing efficiency; therefore, designing and finding suitable target sites is crucial before undertaking gene editing. After designing specific target sites, in vitro cell activity screening is also necessary to obtain effective target sites for subsequent experiments.

[0064] As one embodiment of the present invention, virus-induced gene silencing (VIGS) can be used to suppress SPD6, thereby improving the plant's disease resistance.

[0065] It should be understood that those skilled in the art, upon learning of the correlation between SPD6 and plant traits, can prepare molecules that downregulate SPD6 through various pathways, thereby enabling the regulation of plant traits. These regulatory molecules can be delivered into plants via transgenic technology, or through various techniques known in the art, such as hybridization.

[0066] This invention also relates to using SPD6 as a tracking marker for the progeny of gene-transformed plants. Furthermore, this invention relates to using SPD6 as a molecular marker to determine the disease resistance of plants by detecting the expression of SPD6 in them.

[0067] It should be understood that although the SPD6 of the present invention is preferably obtained from rice, other genes obtained from other plants that are highly homologous to rice SPD6 (e.g., having more than 80%, such as 85%, 90%, 95%, or even 98% sequence identity) are also within the scope of the present invention. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0068] After learning about the mechanism by which SPD6 promotes plant disease resistance, we can use this new discovery to screen for substances or potential substances that can regulate plant disease resistance by modulating SPD6.

[0069] Therefore, the present invention provides a method for screening reagents that improve the disease resistance of plants, the method comprising: (1) adding the test substance to a system containing SPD6; (2) observing the expression or activity of SPD6 in the system of (1); if the test substance inhibits the expression or activity of SPD6, it indicates that the test substance is a reagent for improving the disease resistance of grass plants.

[0070] Methods for screening substances that act on proteins or specific regions thereof as targets are well known to those skilled in the art, and these methods can all be used in this invention. The candidate substances can be selected from: peptides, polymeric peptides, peptide-like substances, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, and nucleic acid sequences, etc. Depending on the type of substance to be screened, those skilled in the art will understand how to select an appropriate screening method.

[0071] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Science Press, or according to the manufacturer's recommendations.

[0072] Experimental materials

[0073] Wild-type rice variety: Japonica rice Nipponbare (NIP).

[0074] Rice blast fungus (M. oryzae): TH12, GUY11, YN2 (isolated from rice).

[0075] Bacterium oxysporum (Xoo): PXO99A, J18 (DY89031).

[0076] The transgenic lines (using Nipponbare as wild-type material) were established as follows:

[0077] Establishment of SPD6-OE / NIP:

[0078] Using pUN1301 preserved in our laboratory as the vector backbone, driven by the maize Ubiquitin promoter, the SPD6 CDS sequence was linked, and the N-terminus was linked with the GFP sequence as a marker to construct an SPD6 overexpression vector. The vector was then transformed into NIP callus to obtain rice transgenic material that overexpresses SPD6 in the NIP background, namely SPD6-OE / NIP.

[0079] The establishment of SPD6-KO / NIP:

[0080] In the NIP context, the target sequence was located at the 36bp to 55bp site of the third exon of SPD6, which was linked to the osu6b-sgRNA expression cassette and constructed into the CRSPR / CAS9 vector. Rice callus was then transformed to obtain the SPD6 knockout transgenic material, namely SPD6-KO.

[0081] Experimental methods

[0082] 1. Inoculation with rice blast fungus

[0083] 1.1 Field injection inoculation with rice blast fungus

[0084] The rice blast fungus was activated by inoculating races onto CM medium and incubating at 28°C (12h light / 12h dark) for 10–12 days. The medium was then rinsed with 0.05% Tween 20 sterile water, and the spore suspension was washed off with a glass rod, filtered through a 40μm membrane, and finally adjusted to a concentration of 1×10⁻⁶. 5 The spore count was 30 to 50 spores per field of view under a 10×10x microscope, and was used for inoculation. Approximately 4 weeks after rice transplanting, the spore solution was injected using a syringe about 2 cm below the ligule of newly emerging leaves until it overflowed from the tips of the new leaves. Disease incidence could be observed 7 days after inoculation for rice blast.

[0085] 1.2 In vitro inoculation with rice blast fungus

[0086] (1) Prepare 0.7% Agar using ddH2O, heat to melt and pour into a 10cm square dish, about 6 pieces / 200mL.

[0087] (2) Take tender leaves that have grown in the field or greenhouse for one month, cut 9cm leaves and place them in a square dish. Cover the top and bottom ends with absorbent paper soaked with 6-BA solution at a concentration of 1μg / ml to keep the leaves moist and prevent yellowing.

[0088] (3) Use a 1ml syringe needle to scrape the surface of the leaf to create a wound (be careful not to penetrate the leaf), and select 3 points evenly on each leaf.

[0089] (4) Wash 1×10 5 Rice blast fungus spore solution with spores / mL, 10μL of spore solution was taken and applied to the wound on the leaf to form droplets.

[0090] (5) Observe the phenotype and measure the length of the lesion in a 28℃ incubator, in darkness for 4 hours, followed by 12 hours of light and 12 hours of darkness. Observe the phenotype and measure the length of the lesion in about 5-7 days.

[0091] 1.3 Statistics on the growth of rice blast

[0092] (1) Take a certain amount of rice leaves infected with rice blast and crush them with liquid nitrogen.

[0093] (2) Add 400 μL of extraction buffer (50 mM Tris-Cl, pH 7.5; 20 mM EDTANa2, pH 8.0; 2% Sarcosine; 0.5% SDS; 5 M Urea, 5% Phenol, 0.3 M NaCl) and shake to mix.

[0094] (3) Add 400 μL of phenol / chloroform / isoamyl alcohol in a 25:24:1 ratio and shake vigorously for 10 min.

[0095] (4) After centrifuging at 12000 rpm for 10 min, transfer the supernatant to a new 1.5 ml EP tube. Add an equal volume of isopropanol and precipitate at -20℃ for 10 min.

[0096] (5) Centrifuge at 12000 rpm for 10 min and discard the supernatant. Add 1 mL of 75% ethanol to wash the precipitate, centrifuge at 7500 rpm for 5 min, remove the supernatant, and dry at room temperature. Add 40 μL of ddH2O (containing 10 μg / mL RNase) to dissolve the precipitate.

[0097] (6) Perform Real-time PCR to detect the relative content of rice blast fungus DNA.

[0098] 2. Inoculation with bacterial blight pathogen

[0099] 2.1 Activation culture of Blightococcus faecium

[0100] (1) Take the Xoo strain stored at -80℃, streak it on NA (with 15mg / L cephalexin) solid medium to activate it, and incubate it upside down at 28℃ for 72h.

[0101] (2) After the single colony has grown, pick the single colony and put it into NA (with 15 mg / L cephalexin) liquid medium and shake gently for 48 h.

[0102] (3) Take 200 μL of small-shake bacterial solution into NA solid medium, spread it evenly with a sterilized spreader until the bacterial solution is dry, and incubate at 28℃ upside down for 48 h. The white leaf blight pathogen that grows can be used to inoculate rice.

[0103] 2.2 Leaf-cutting method for field inoculation with pathogens

[0104] Scrape off the healthy white leaf blight fungus grown on the NA medium with sterile water and dilute to OD=1.0. Using scissors, take a 2cm section from the tip of a rice leaf (approximately 60 days old) and cut it obliquely downwards for inoculation. After approximately 14 days, measure the length of the leaf lesions.

[0105] 3. Transformation of callus tissue in mature rice embryos

[0106] 3.1 Induction of callus from mature rice embryos

[0107] (1) Use a threshing machine to remove the husks from the rice seeds and discard any moldy or deformed seeds.

[0108] (2) Add a small amount of 75% ethanol, shake by hand for about 30 seconds, and rinse once with sterile water. Add 25-30% (v / v) NaClO, and shake on a shaker at 200 rpm for 30 minutes.

[0109] (3) Rinse with sterile water 5-6 times, and once in between, place it in a shaker and shake for 10 minutes.

[0110] (4) Place the seeds on sterilized filter paper to absorb the moisture on the seed surface and induce callus formation.

[0111] (5) After culturing in the dark for about 14 days, the endosperm, plumule and embryonic root are removed. The resulting callus can be used for transgenic or subculture. Subculture is performed every two weeks. The number of subcultures depends on the state of the callus.

[0112] 3.2 Preparation of Agrobacterium-mediated transformation culture

[0113] (1) The constructed plasmid was transferred into EHA105 competent cells and cultured at 28°C for two days.

[0114] (2) Select a single clone and place it in 5 mL of LB liquid medium containing the corresponding antibiotic for culture. Culture at 28°C with shaking for 48 h.

[0115] (3) Take 1 mL of the overnight culture and transfer it into 15 mL of AB (20 mg / L Rif + 50 mg / L Kan + 100 mg / L AS) liquid culture medium. Incubate at 28°C until OD600 = 0.5 (about 4 h).

[0116] 3.3 Co-culture of rice callus and bacterial solution

[0117] (1) Centrifuge the bacterial culture at 5000 rpm for 10 min and discard the supernatant.

[0118] (2) Resuspend the bacterial cells in AAM containing 100 mg / L AS until the bacterial solution OD600 = 0.4-0.6.

[0119] (3) Co-culture the bacterial solution with rice callus for 20 min.

[0120] (4) Blot dry the bacterial solution, pick up the callus tissue and place it on NBD solid culture medium (with 100 mg / L AS) with sterile filter paper on it. Add 1 mL of AAM (with 100 mg / L AS) culture medium to each dish to fully moisten the sterile filter paper. Incubate for 2-3 days.

[0121] 3.4 Screening

[0122] Blot the callus tissue dry with sterile filter paper and transfer it to a selection medium containing hygromycin to screen for resistant callus. Change the medium every two weeks.

[0123] 3.5 Differentiation

[0124] Selected rice callus tissues were transferred to rice differentiation medium and cultured under light. The medium was changed every two weeks until the callus differentiated into seedlings.

[0125] 3.6 Rooting

[0126] Transfer the seedlings from the differentiation medium to the rooting medium. After about 2 weeks of growth, remove the seedlings, wash off the agar medium, and culture them in water for 7 days before transplanting them into the soil.

[0127] 4. DAB staining to detect H2O2 accumulation

[0128] (1) Take rice leaves and immerse them in DAB staining solution. Vacuum can be drawn to allow the staining solution to enter the leaf tissue.

[0129] (2) Reaction under light at room temperature for 8-12 hours.

[0130] (3) After the reaction is complete, take out the leaves and immerse them in 95% ethanol solution, decolorize in boiling water bath for 10 min, and decolorize in 95% ethanol solution for more than 4 h until decolorization is complete.

[0131] (4) Observe whether brown spots appear on the leaves (the polymer formed by the reaction of DAB and H2O2 is brown).

[0132] Example 1: Identification and Expression Characteristics of SPD6

[0133] The inventors discovered a dwarfing mutant in a Nipponbare (NIP) varietal background during field experiments, exhibiting a lesion-like phenotype on its leaf surface. Sequencing analysis revealed a mutation in the SPD6 gene in the mutant material; SPD6 encodes a leucine carboxymethyltransferase.

[0134] Confocal observations were performed on the root tips of SPD6-OE, a GFP-tagged SPD6 overexpression material in tobacco and rice. The results showed that SPD6 was expressed in both the plant cell nucleus and cell membrane. Figure 2 A).

[0135] Furthermore, a luciferase complementation experiment was conducted to verify this. The results showed that SPD6 itself exhibits interactions ( Figure 2 B).

[0136] Spd6 genomic DNA sequence (SEQ ID NO:1):

[0137] ATCACGTCTCTCGTCTTCCTCTCCCTCTCACGCCGTGACCCAGCGATCGACCGGCCGGAATG

[0138] GATGCGGCGGCGGCGGCGGCGGCGGCGGGAGGAGGAGGAGGGGGAGGCGGTAGCGTCGCG

[0139] GCGCGGAGCAGCCCGGCGTCCGTGCAGGCCACCAACGACGACGCCGCGGCCAGCAAGCTGT

[0140] GAGTGACCAACCCCGACTCCCCCCCTCTACCTCTCCAGCTCTCCTTCCCCTCGCGTAGTCGCA

[0141] TCCGCATGACCACCAGGTCTCGTGTGCTCCGCGTGATATTCTCTCCTTTGCACGAGGCGCCAT

[0142] CGGGAAGCGGTCGCCCCTGTGCTTGTTTGGATCAATCCGCGGTCGCGCTATGCTGCTACTGT

[0143] TTGTTGCGCTAGTGAATACCCTGACAAAATGGAATACCGTGACAAAATGAAGCCAAAGCCA

[0144] TGCTGAATCGTGATGCTAAAATGCAATTATTGTACTAAGCAAGAGGGATGGTTTATACAGTT

[0145] TTTATGTGAAGATCCAGAATCGCGCTCCACTTAGGTGACCCTTTTCTGCGTAGGACCATGCA

[0146] ATGTCGTATAACCACTCCTTTGAACTGATTCTATTTTTATTTCTCTCTGCAATAAATCAGTGT

[0147] GTAATGGTAGGAAAAGAGCTATGTGCCTCTTTTACCAATTGCCTTGAATGAAGTCCTTCTGT

[0148] TGAGTGGATCAGCTAGCTTGCAACTTTATATATTCTTGTGTGTTTCAGAAGAAATTTCTTGAT

[0149] ATTTTCTTGCTTATTTGTTTGGAGTGAATTGATGCTTTAAGTACTGGTTTGAAGAAATTGATT

[0150] GGGCATCTCAACTTCCAGTGCTTATCTGGCCTACTGACCTTGGTATGCAGAGCAAATGATCT

[0151] ACCTTCACCTCTTCTGCCTATGACATTGCAGAGGCCGCTATCTTGAACAATGAACAGATAAA

[0152] TGTAACACGGTCCATGTTATTCAATATGCATGGAAAGTGTGGTGTATTGCTTATACATATAG

[0153] ATGTCATTGTAGTTTTCAAAGTAGAGGTTGACTGTTGCTGCTTGCTATATGCCATACTCATTT

[0154] TCATTTTTCCTTTGCAGGTCATGTGTTAATAAAGGATACATGAAGGATGATTATGTGCACTTC

[0155] TTTGTCAGACGAACAACAAAAAGGGCTCCAATAATAAATCGAGGTTGGTAGTGCCTCATTGA

[0156] ATCTTTATCTGCTTCTCTAAAATTGAAAATGTTTGAAAATTCTAATTTGTTTACCTTTCCAAA

[0157] TAACTCTGCAAAGGGTACTATGCACGTTGGTCTGTTCTTAGGAAGCTTCTGCATCAGTTCCTC

[0158] GGTGCTGGAAATGGCAGTAATGATCAAAACCGGAAACAAATATTATCTCTTGGTGCTGGCTT

[0159] TGACACGACATTCTTCCAGTTGCAGGTTATCCATTACTCACTATTCATGAGCATCACAATCAC

[0160] ATGCTTACGGCCCATTATCAGATCTACTGTTTGATCTGAATAATTTATATCATTTTTATTTAA

[0161] CTCTGTATGCCCTTTGTTACTATAAACTAGTTAAATAGGTTCCAGTACGCCTTGCATCTTAAT

[0162] CATATCCTGCTTTGGTGGAGGCAGTGTTTAAGATCAAACTTCACAAAGTATATTGGTATATT

[0163] TTTAATATCAATTCATCTCTCTCAGTCTGTGCTGTATTTGTTACTGTAGGACGAAGGCATTGC

[0164] TCCATACCTTTATGTTGAGTTGGATTTCAAGGAGGTAGATAAAGTTTCTTTGCTATTTTCAAG

[0165] TGTAGCATAGTACTCTGCATAATGTGATCATGTAATGGCAGCGGAGCAGCTTGTTCTCTTT

[0166] GTGCATAAGTAACTTTTCTGGCAACTTTTGATGATTGGCAGGTAACCAGCAAAAGGCTGCC

[0167] ATCATTAACCACTATAGTCAAATGAAGGAAGTTAGGACCAGAAGCTTCGATATCAATTG

[0168] GTAGTCTTCTCATTTGCATATGATACTGTCGATTAATGCCTACCCTGTTTATATCTAAGGATC

[0169] TTCCCCACTAATGGTTCTACCATGTTCTTTCTATATGTATCTACCGTAATACTTGGTGCCTTTT

[0170] TCTTATATGCATCTTTAGCAGAAATAAGTGCTAATGACAATTAAGGAATGTGTGCTAATTT

[0171] ACTTAGACCCAGCTTCAACCATCTAGAGGCCATTCTCCACATCTAGAGGCTAATTTATTTGTT

[0172] TTTTGTTTGCCTGAATCCATCTAGAGGCCATTCTCCACGTTTATGTTGATTTATTCAGTACCTT

[0173] TTTTGTATAAAGTTCCTGTTATATTTTCTCTTGGAGGTAGCTATTTCATTCTGTTTGTAAGAAT

[0174] GTTTCTCTTGGTATAACTTAATTGACAGCATAATGGTCTTAGTGACCAAAATGATTTATACCC

[0175] TGGCCAGAATTGTGAGTTGAAGTGAACTTTCCATATCACAGAATTTGCTATTTTCTATTTTCC

[0176] GTGTTCTGATATTAATTGATAATGGCCAAAATATTTCAGAAAAAGTGAAGTGGAAGGTGCGC

[0177] ATTACAAGCTCTTTTCTGCTGATATTCGTGACATACCAAAACTTGATTCAGTTATTCAGATGG

[0178]

[0179] CDS sequence (SEQ ID NO:2) of Spd6 gene:

[0180] ATGGATGCGGCGGCGGCGGCGGCGGCGGGAGGAGGAGGAGGGGGAGGCGGTAGCGTC

[0181] GCGGCGCGGAGCAGCCCGGCGTCCGTGCAGGCCACCAACGACGACGCCGCGGCCAGCAAG

[0182] CTGTCATGTGTTAATAAAGGATACATGAAGGATGATTATGTGCACTTCTTTGTCAGACGA

[0183] ACAACAAAAAGGGCTCCAATAATAAATCGAGGGTACTATGCACGTTGGTCTGTTCTTAGG

[0184] AAGCTTCTGCATCAGTTCCTCGGTGCTGGAAATGGCAGTAATGATCAAAACCGGAAACAA

[0185] ATATTATCTCTTGGTGCTGGCTTTGACACGACATTCTTCCAGTTGCAGGACGAAGGCATT

[0186] GCTCCATACCTTTATGTTGAGTTGGATTTCAAGGAGGTAACCAGCAAAAAGGCTGCCATC

[0187] ATTAACCACTATAGTCAAATGAAGGAAAAGTTAGGACCAGAAGCTTCGATATCAATTGAA

[0188] AAAGGTGAAGTGAGAAGTGCGCATTACAAGCTCTTTTCTGCTGATATTCGTGACATACCA

[0189] AAACTTGATTCAGTTATTCAGATGGCTGAAATGGACCCTACCTTGCCGACCTTTATAATT

[0190] GCAGAGTGTGTGCTAATTTACTTAGACCCAGCTTCAACCAGTTCTATTGTTATTTGGGCA

[0191] TCTGATAAGTTCTCCACTGCCATATTTTTCTTATATGAGCAGATCCATCCAGATGATGCA

[0192] TTTGGAGAGCAAATGATTATAAACCTTGAGAGTAGAGGATGCCCTCTCCTTGGTATAAAT

[0193] GCTACACCAACCCTAAGTCACAAGGAAAATCTTTTTCTTGATCACGGATGGCAGAGAGCT

[0194] GTTGCATGGGATATGCTAAAAATATACAATGATTTCATTGACAGTGAAGAAAGACGCAGG

[0195] ATTGAACGATTGGAATTGTTTGATGAGTTCGAAGAGTGGCATATGATGCAGGAACACTAT

[0196] TGTGTGGCTTATGGAATAAATGATGCCAAGGGCCTGTTTGATGATTTTGGGTTTAAGGAC

[0197] TAG

[0198] Protein sequence of Spd6 gene (SEQ ID NO:3):

[0199] MDAAAAAAAAGGGGGGGGSVAARSSPASVQATNDDAAASKLSCVNKGYMKDDYVHFFVRR

[0200] TTKRAPIINRGYYARWSVLRKLLHQFLGAGNGSNDQNRKQILSLGAGFDTTFFQLQDEGI

[0201] APYLYVELDFKEVTSKKAAIINHYSQMKEKLGPEASISIEKGEVRSAHYKLFSADIRDIP

[0202] KLDSVIQMAEMDPTLPTFIIAECVLIYLDPASTSSIVIWASDKFSTAIFFLYEQIHPDDA

[0203] FGEQMIINLESRGCPLLGINATPTLSHKENLFLDHGWQRAVAWDMLKIYNDFIDSEERRR

[0204] IERLELFDEFEEWHMMQEHYCVAYGINDAKGLFDDFGFKD

[0205] Example 2: Obtaining the SPD6 transgenic line

[0206] In the context of NIP, SPD6 knockout transgenic material, namely SPD6-KO, was constructed using the CRSPR / CAS9 gene editing system.

[0207] The sgRNA target sequence is: CTGCATCAGTTCCTCGGTGC (SEQ ID NO:4)

[0208] A series of SPD6 knockout transgenic lines were obtained. Among them, SPD6-KO#1 had a 1bp insertion mutation between positions 52bp and 53bp in exon 3, resulting in frameshift and premature termination of protein expression; SPD6-KO#1 had a four-base deletion mutation between positions 53bp and 56bp in exon 3, resulting in premature termination of protein expression. Figure 1 A).

[0209] Meanwhile, the material SPD-OE, overexpressed in the NIP context, was constructed using the following primers:

[0210] pUbi-SPD6-GFP-F:

[0211] caggtcgactctagaggatccATGGATGCGGCGGCGGCG (SEQ ID NO: 5);

[0212] pUbi-SPD6-GFP-R:

[0213] ctcgcccttgctcacggtaccGTCCTTAAACCCAAAATCATCAAAC (SEQ ID NO: 6).

[0214] Expression analysis was performed on the obtained SPD6-OE and SPD6-KO materials, and the results are as follows: Figure 1 B indicates that transgenic lines with up- or down-regulated SPD6 gene were successfully obtained.

[0215] SPD6-KO exhibits an autoimmune phenotype, with lesion-like spots appearing on the leaf surface. DAB staining revealed significantly higher H2O2 accumulation in SPD6-KO material compared to NIP and SPD6-OE. Figure 1D). These results suggest that SPD6 is involved in regulating plant immunity.

[0216] Example 3: SPD6 negatively regulates rice disease resistance

[0217] To investigate whether SPD6 can regulate rice immunity, the transgenic materials obtained were used with NIP as a wild-type control. Leaves were pruned in the field and inoculated with bacterial blight pathogens PXO99A and J18. The length of lesions was measured 14 days later. Figure 3 ).

[0218] Statistical results showed that, compared with the wild-type NIP, the lesion length of the SPD6-KO material was significantly shorter, indicating a significantly enhanced resistance to bacterial blight. The lesion length of the SPD6-OE material was slightly longer, but the difference was not significant.

[0219] The above results indicate that SPD6 negatively modulates rice bacterial blight resistance in the NIP background, and the SPD6-KO material significantly enhances resistance to bacterial blight.

[0220] Example 3: SPD6 regulates rice blast resistance

[0221] Further research is needed to determine whether SPD6 can regulate rice blast resistance.

[0222] 1. Resistance to rice blast race TH12

[0223] Using NIP as a wild-type control, materials overexpressing and knocked out SPD6 were inoculated with NIP-in vitro for the toxic rice blast race TH12.

[0224] The results showed that the SPD6-KO line had fewer blast lesions than the NIP line, indicating greater resistance; while the SPD6-OE line was more susceptible to the disease. Figure 4 A, B).

[0225] The growth of rice blast fungus was calculated by qRT-PCR using rice Ubiquitin as an internal control against the housekeeping gene MoPOT2 of the leaf blast fungus.

[0226] The results showed that the bacterial growth in SPD6-KO material after inoculation was significantly lower than that on NIP; conversely, the growth of rice blast fungus in SPD6-OE material was significantly higher than that on NIP. Figure 4 A, B).

[0227] 2. Resistance to rice blast race Guy11

[0228] The SPD6-OE and SPD6-KO materials were inoculated with Guy11, a rice blast race with relatively low virulence compared to TH12, through in vitro inoculation.

[0229] The results showed that the disease phenotype after TH12 vaccination was consistent with that after TH12 vaccination, further validating the role of SPD6 in regulating baseline disease resistance. Figure 4 C).

[0230] 3. Resistance to rice blast fungus race YN2

[0231] To verify whether SPD6 is involved in regulating the resistance mediated by the rice blast resistance gene Pish within NIP, the inventors inoculated the NIP resistant race YN2.

[0232] The results showed that Pish-mediated disease resistance was reduced in SPD6-OE materials, and the bacterial growth was significantly higher than that in NIP; SPD6-KO materials, on the other hand, showed greater disease resistance, suggesting that SPD6 may be involved in regulating the ETI immune response process. Figure 4 D).

[0233] These results all indicate that SPD6 can negatively regulate rice blast resistance.

[0234] discuss

[0235] In summary, SPD6 can negatively regulate resistance to rice bacterial blight and rice blast, and the SPD6-KO material exhibits enhanced disease resistance. Further research on SPD6 can contribute to a more comprehensive understanding of the regulatory mechanisms of disease resistance and susceptibility, and is of great significance for maintaining the balance between growth and immunity, providing new insights for rice molecular breeding.

[0236] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. A method for improving the disease resistance of grass plants, the method comprising downregulating SPD6 in grass plants.

2. The method as described in claim 1, characterized in that, The downregulation of SPD6 includes: knocking out or silencing the SPD6 gene in plants, or inhibiting the activity of the SPD6 protein; preferably, it includes: knocking out the SPD6 gene using gene editing methods, silencing SPD6 using interfering molecules that specifically interfere with SPD6 gene expression, knocking out the SPD6 gene using homologous recombination methods, or inhibiting SPD6 expression by ultraviolet stress; preferably, knocking out the SPD6 gene using gene editing methods, targeting its third exon; more preferably, causing a 1 bp insertion mutation between the 52 bp and 53 bp sites of the third exon or a four-base deletion mutation between the 53 bp and 56 bp sites of the third exon; more preferably, gene editing is performed using sgRNA with the nucleotide sequence shown in SEQ ID NO:

4.

3. The method as described in claim 1, characterized in that, The disease resistance mentioned refers to the plant's ability to resist pathogens, including fungi or bacteria; Preferably, the fungus includes: rice blast fungus; Preferably, the bacteria include Xanthomonas.

4. The method as described in claim 1, characterized in that, The SPD6 is selected from: (a) a protein with the amino acid sequence of SEQ ID NO:3; (b) a protein derived from (a) that has the function of the protein in (a) formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence of SEQ ID NO:3; or (c) a protein derived from (a) that has more than 80% homology with the protein sequence defined in (a) and has the function of the protein in (a); or (d) a protein formed by adding a tag sequence to the N or C end of the protein in (a), (b) or (c), or by adding a signal peptide sequence to the N end of the protein; preferably, the SPD6 includes a protein encoded by SEQ ID NO:1 or SEQ ID NO:

2.

5. An application of SPD6 for use as a downregulation target to enhance the disease resistance of grasses; or for screening reagents that target SPD6 to enhance the disease resistance of grasses.

6. The use of an SPD6 conditioning agent for improving the disease resistance of gramineous plants; preferably, the SPD6 conditioning agent comprises: Down-regulatory agents that knock out or silence the SPD6 gene or inhibit the activity of the SPD6 protein.

7. The use as described in claim 6, characterized in that, The SPD6 down-regulatory agent comprises: a gene editing reagent for knocking out the SPD6 gene, an interfering molecule that specifically interferes with the expression of the SPD6 gene, and a reagent for knocking out the SPD6 gene based on homologous recombination; more preferably, the SPD6 down-regulatory agent comprises a gene editing reagent targeting the third exon of the SPD6 gene; more preferably, the gene editing reagent is sgRNA with the nucleotide sequence shown in SEQ ID NO:4; or The disease resistance mentioned refers to the plant's ability to resist pathogens, including fungi or bacteria; preferably, the fungi include rice blast fungus; preferably, the bacteria include bacterial blight fungus.

8. The method as described in any one of claims 1 to 5 or the use as described in any one of claims 6 to 7, characterized in that, The grasses include cereal plants, or the SPD6 is derived from cereal plants; preferably, the grasses include: rice, wheat, millet, foxtail millet, corn, sorghum, millet, barley, rye, oats, and short-stalked grass.

9. A method for screening reagents to enhance the disease resistance of gramineous plants, the method comprising: (1) Add the analyte to a system containing SPD6; (2) Observe the expression or activity of SPD6 in the system of (1); if the analyte inhibits the expression or activity of SPD6, it indicates that the analyte is a reagent to improve the disease resistance of grass plants.

10. An application of SPD6 as a molecular marker for identifying disease resistance in grasses.

11. A method for specifically selecting plants with enhanced disease resistance, the method comprising: To identify the expression of SPD6 in the test plant, if the SPD6 expression of the test plant is significantly lower than the average SPD6 expression value of this type of plant, then it is a plant with enhanced disease resistance.

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