Use of HPS1 gene or protein encoded by HPS1 gene in regulation of plant resistance to diseases and pests

Abstract

Use of HPS1 or a protein encoded by the HPS1 gene in the regulation of plant resistance to diseases. A comprehensive appraisal is performed on the resistance function of the rice transcription factor gene, i.e. HPS1 gene, to rice biotic stresses by means of using a series of approaches such as genetics, molecular biology and pathology. It is found that the rice overexpressing the HPS1 gene has stronger resistance to different fungal or bacterial diseases, such as rice blast, sheath blight and bacterial leaf blight, and has stronger resistance to pest damage caused by Nilaparvata lugens compared to a wild-type rice plant. The rice with the knockout of the HPS1 gene that causes the protein-encoding ability of the HPS1 gene to be blocked has weaker resistance to biotic stress compared to the wild-type rice. Therefore, the HPS1 gene provides an important theoretical basis for selecting and breeding crop varieties with relatively high resistance to biotic stress.

Description

Application of HPS1 gene or protein encoded by HPS1 gene in regulating plant disease and insect pest resistance Technical Field

[0001] The present invention relates to the field of crop breeding, and in particular to the application of HPS1 gene or protein encoded by HPS1 gene in regulating the disease and insect pest resistance of plants. Background Art

[0002] Rice is one of the world's most important food crops, serving as the staple food for over half of the world's population. It is also the largest cultivated crop in my country, and therefore its yield is crucial to the country's food security. Biological stresses, particularly diseases and insect pests, pose a serious threat to rice production. Currently, chemical control is the primary method for controlling rice diseases and insect pests. However, these methods often only address the symptoms, not the root causes. This not only leads to the development of pesticide resistance in pathogens and pests, but also creates a host of concerning issues, including environmental pollution, ecological crises, food security, and health risks.

[0003] Therefore, identifying and utilizing genes and mechanisms that contribute to biotic stress resistance is the most effective and environmentally friendly approach to crop improvement. Transcription factors are proteins with DNA-binding domains that bind to specific cis-acting elements in gene promoter regions to regulate target gene expression. They are widely involved in regulating plant resistance to biotic stresses. At least 167 bHLH family transcription factor genes have been identified in rice, but few studies have examined the functions of these transcription factors in regulating biotic stress resistance. Therefore, identifying bHLH transcription factors that regulate biotic stress resistance and utilizing them to improve crop resistance has significant application value. Summary of the Invention

[0004] The present invention aims to provide the use of the HPS1 gene or the protein encoded by the HPS1 gene in regulating plant disease and pest resistance, thereby overcoming the problems of the prior art. The present invention is the first to discover the use of the HPS1 gene in regulating plant disease and pest resistance, and overexpressing the gene can enhance plant disease and pest resistance.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an application of an HPS1 gene or a protein encoded by the HPS1 gene in regulating plant disease resistance. The nucleotide sequence of the HPS1 gene is shown in SEQ ID NO.1; the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0007] Preferably, the regulation includes increasing plant disease resistance and decreasing plant disease resistance.

[0008] Preferably, by knocking out the HPS1 gene, the disease resistance of the plant is reduced; by overexpressing the HPS1 gene, the disease resistance of the plant is improved.

[0009] Preferably, the disease resistance includes the ability to resist diseases caused by fungi and the ability to resist diseases caused by bacteria.

[0010] Preferably, the fungi include pathogens that cause rice blast and / or pathogens that cause sheath blight;

[0011] The bacteria include pathogenic bacteria that cause bacterial blight.

[0012] Preferably, the plant comprises rice.

[0013] The present invention provides the use of the HPS1 gene or the protein encoded by the HPS1 gene in regulating the insect pest resistance of plants. The nucleotide sequence of the HPS1 gene is shown in SEQ ID NO.1; the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0014] Preferably, the regulation includes increasing the plant's resistance to insect pests and decreasing the plant's resistance to insect pests.

[0015] Preferably, by knocking out the HPS1 gene, the insect pest resistance of the plant is reduced; by overexpressing the HPS1 gene, the insect pest resistance of the plant is improved.

[0016] Preferably, the plant comprises rice.

[0017] The present invention discloses the following technical effects:

[0018] The present invention provides the use of a rice gene encoding a bHLH transcription factor, hydrogen peroxide sensor 1 (HPS1), or a protein encoded by the HPS1 gene, in regulating plant disease and insect pest resistance. The present invention comprehensively characterizes the rice transcription factor gene HPS1's function in resisting biotic stresses in rice using a range of methods, including genetics, molecular biology, and pathology. The present invention found that rice plants overexpressing the HPS1 gene exhibited greater resistance to various fungal and bacterial diseases, such as rice blast, sheath blight, and bacterial leaf blight, than wild-type rice plants, as well as greater resistance to pests caused by brown planthoppers. Furthermore, rice plants in which the HPS1 gene's ability to encode the protein was impaired by knockout showed weaker resistance to biotic stresses than wild-type rice. Therefore, the present invention identifies a key gene in rice that regulates resistance to multiple biotic stresses. The HPS1 gene provides a significant theoretical basis for breeding crop varieties with enhanced resistance to biotic stresses.

[0019] In a specific embodiment, the present invention successfully obtained two independent HPS1 gene function loss knockout strains through gene editing; by transferring the overexpression plasmid into rice through Agrobacterium transformation, two independent HPS1 gene overexpression rice strains were successfully obtained; and at the same time, an HPS1 gene overexpression strain was obtained. The present invention explores the regulatory effect of the HPS1 gene on crop biotic stress resistance through phenotypic identification of these transgenic strains. The regulatory effect of the HPS1 gene on crop biotic stress resistance is specifically as follows: after overexpressing the HPS1 gene, rice resistance to different fungal or bacterial diseases such as rice blast, sheath blight and white leaf blight can be enhanced, as well as resistance to pests caused by brown planthoppers, while knocking out the HPS1 gene weakens rice resistance to biotic stresses such as these diseases and pests. Therefore, the HPS1 gene can be used to improve the biotic stress resistance of different crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 is a schematic diagram of the construction of HPS1 gene knockout (HPS1-KO) and HPS1 gene overexpression (HPS1-OE) rice plants. A is a schematic diagram of two independent target sites designed for HPS1 gene knockout using the CRISPR / Cas9 system. B is a comparison of the knockout target sequences in two independent HPS1-KO transgenic lines (HPS1-KO#1 and HPS1-KO#6) and Kitaake plants obtained by PCR sequencing. C is an alignment of the predicted amino acid sequences of HPS1 proteins in Kitaake, HPS1-KO#1, and HPS1-KO#6 plants. Figure 2. D shows the expression of HPS1 in Kitaake and different HPS1-OE plants (mean ± sd, n = 3 samples). The data were analyzed using a two-tailed Student's t-test. Specific P values ​​are shown in the figure. P < 0.05 indicates a significant difference, and P < 0.01 indicates an extremely significant difference. E shows the accumulation of HPS1 protein in HPS1-OE plants by immunoblotting analysis. The arrow indicates the HPS1-YFP protein obtained by immunoprecipitation (IP) of total protein extracted from rice leaves using GFP-trap agarose.

[0022] Figure 2 shows the identification of the HPS1 gene's function in regulating rice blast resistance. A shows the leaves of three-week-old Kitaake, HPS1-KO, and HPS1-OE plants inoculated with blast fungus (physiological subspecies Zhong10-8-14). The figure shows photos of representative lesions on rice leaves 7 days after inoculation and statistics of lesion length (mean ± sd, n = 9 lesions). The scale bar is 1 cm. B shows the HPS1 gene's enhanced resistance to blast fungus in rice in the field. Kitaake, HPS1-KO, and HPS1-OE plants aged 18 years were spray-inoculated with the blast fungus (physiological subspecies 10-8-14) in the field. The figure shows photographs of typical lesions on rice leaves seven days after spray inoculation and the number of lesions per leaf (mean ± SD, n ≥ 7 leaves). Data were analyzed using a two-tailed Student's t-test. Specific P values ​​are indicated in the figures; P < 0.05 indicates a significant difference, and P < 0.01 indicates an extremely significant difference. Scale bar, 2 cm.

[0023] Figure 3 shows the identification of the HPS1 gene's function in regulating rice resistance to sheath blight. The figure shows photographs of typical lesions on leaves of three-week-old Kitaake, HPS1-KO, and HPS1-OE plants two days after inoculation with sheath blight (physiological race AG-1-IA) and statistics of lesion length (mean ± sd, n = 10 lesions). The scale bar is 1 cm. Data were analyzed using a two-tailed Student's t-test. Specific P values ​​are shown in the figure; P < 0.05 indicates a significant difference, and P < 0.01 indicates an extremely significant difference.

[0024] Figure 4 illustrates the identification of the HPS1 gene's role in regulating rice resistance to bacterial blight. The figure shows representative photographs of lesions on leaves of three-week-old Kitaake, HPS1-KO, and HPS1-OE plants 14 days after inoculation with bacterial blight (race PXO99A) and statistical lesion lengths (mean ± SD, n = 10 lesions). Data were analyzed using a two-tailed Student's t-test. Specific P values ​​are indicated in the figure; P < 0.05 indicates significant differences, and P < 0.01 indicates extremely significant differences. Scale bar, 1 cm.

[0025] Figure 5 shows the identification of the HPS1 gene's function in regulating rice resistance to brown planthoppers. The figure shows the phenotypes and BPH resistance scores of two-leaf-stage Kitaake, HPS1-KO, and HPS1-OE seedlings four days after being fed by brown planthoppers (mean ± SD, n = 30 samples). Data were analyzed using a two-tailed Student's t-test. Specific P values ​​are shown in the figure; P < 0.05 indicates a significant difference, and P < 0.01 indicates an extremely significant difference.

[0026] FIG6 is a schematic diagram of the work of improving rice resistance to biotic stress using the HPS1 gene. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] The commercially available pCRISPR and pCAMBIA1300 vectors and the published rice blast fungus (physiological race Zhong10-8-14), sheath blight (physiological race AG-1-IA), bacterial leaf blight (physiological race PXO99A), brown planthopper (collected in the greenhouse of Sichuan Agricultural University in 2021) and rice materials (wild type Kitaake, HPS1-KO and HPS1-OE) used in the following examples were provided and preserved by the State Key Laboratory of Southwest Gene Resources Exploration and Utilization, Sichuan Agricultural University.

[0033] Example 1 Construction of HPS1 gene knockout mutant rice plants

[0034] In order to identify the function of the HPS1 gene (ID: Os01g0196300), the inventors used the CRISPR / Cas9 system to design two knockout target sites (Target site 1 and Target site 2) for the HPS1 gene (SEQ ID NO. 1) in wild-type Kitaake plants for gene editing. The nucleotide sequence of Target site 1 is shown in SEQ ID NO. 13, specifically CCGGCAACAACGGCTTCATG, and the nucleotide sequence of Target site 2 is shown in SEQ ID NO. 14, specifically GGCGGGGTTCCATTACCCGG (A in Figure 1).

[0035] The genomic sequence of the HPS1 gene in rice (SEQ ID NO. 1):

[0036] >NC_029256.1:5201837-5203986Oryza sativa Japonica Group cultivar Nipponbare chromosome 1,IRGSP-1.0:

[0037] The inventors synthesized primers with the designed target sequence and annealed the "pCRISPR-HPS1-Target1-KO" primer and the "pCRISPR-HPS1-Target2-KO" primer (Table 1) in a PCR instrument (95°C for 3 min, 0.2°C / sec to 20°C). The pCRISPR plasmid and the annealed primers were then connected using a vector exonuclease linearization-ligation reaction system (Table 2). Agrobacterium carrying the constructed knockout vector was transformed into the callus tissue of wild-type rice Kitaake to obtain transgenic rice plants (the method is referenced in W. Li, et al., A Natural Allele of a Transcription Factor in Rice Confers Broad-Spectrum Blast Resistance. Cell 170(1):14-126,2017).

[0038] Table 1 Primer sequences for detecting HPS1 knockout plants

[0039] Table 2 Vector exonuclease linearization-ligation reaction system

[0040] Next, the inventors used PCR reactions (Tables 3 and 4) and "HPS1-KO-target-1-detection" primers (Table 1) to detect changes in Target 1 in transgenic plants, and PCR reactions and "HPS1-KO-target-2-detection" primers (Table 1) to detect changes in Target 2 in transgenic plants. The results showed that the HPS1 knockout (HPS1-KO) line #1 had a T insertion in Target 1, while another HPS1-KO line #6 had a GTAAT deletion in Target 1 (Figure 1, B). These experiments demonstrate that the inventors have successfully generated rice plants with the HPS1 gene knockout.

[0041] Table 3 PCR reaction system

[0042] The above samples were mixed and centrifuged before PCR reaction. PCR amplification was performed using touchdown PCR. The reaction procedure is shown in Table 4.

[0043] Table 4 PCR reaction procedure

[0044] Further analysis revealed that each mutation in HPS1-KO#1 and HPS1-KO#6 resulted in a frameshift and premature termination of the HPS1 protein (SEQ ID NOs. 2-4 and Figure 1C). Therefore, these results demonstrate that the inventors have successfully generated two independent HPS1 gene loss-of-function knockout lines.

[0045] Amino acid sequence of HPS1 protein in rice (SEQ ID NO. 2):

[0046] MEDCSSWIHGYANANATAGNNGFMCGYAASCSPVEFQQQQQLVGSQIEHHLNQISMQMGMDDESAVYDGASMVDVLLMASSSPHHHAGAGSFQYSSPTSSSASFRSASVSCSPE SSAAATTHFLGPPAPSAAAAGFHYPEVSSQAPLPLPLPPYEPQHGQYTTVLSPPPPAPELPATTTPATGGAFRRYARHLRPRRLPKPGGCGQRMFKTAMSVLTKMHVAATYNRQ YYYQQAAAAAASASAAEAPPSGNQLQHMISERKRREKLNDSFLALKAVLPPGSKKDKTSILIRAREYVKSLESKLSELEEKNRELEARLASRPAAAAKNDKGETAAAPAPEAGDETKRKDLVEIEVTTSGGGAGAADAAAAAGGDQETCTLNVDLRGGGGGGGMSTTDVVLRTLQCLREQIGDGASLVAMSTSAGSGGRPPRANLTLQLKV*, where “*” means a stop codon;

[0047] The amino acid sequence of the HPS1 protein in the gene-edited HPS1-KO#1 rice (SEQ ID NO. 3):

[0048] MEDCSSWIHGYANANATAEQQRLHVRLRCQLQPSRVSAAATAGRLAD*, where “*” means the stop codon;

[0049] The amino acid sequence of the HPS1 protein in HPS1-KO#6 rice after gene editing (SEQ ID NO. 4):

[0050] MEDCSSWIHGYANANATAGNNGFMCGYAASCSPVEFQQQQQLVGSQIEHHLNQISMQMGMDDESAVYDGASMVDVLLMASSSPHHHAGAGSFQYSSPTSSSASFRSASVSCSPESSAAATTHFLGPPAPSAAAAGFPGGLLAGAVATTLAALRAAARPIHHRPLAAAAGARVAGDYYAGDRRRVQAVRAAPPPEEAAQAGRVRAEDVQDGHVGAHQDARGGDVQPPVLLPAGGSRRRVGVGGRGAAVRQPAAAHDLGAEAAGEAQRQLPRPQGRPPSRL*, wherein “*” means a stop codon.

[0051] Example 2 Construction of HPS1 gene overexpressing rice plants

[0052] The inventors amplified the HPS1 coding region sequence using the "pCAMBIA1300-35S:HPS1-OE" primers (Table 5) via PCR (Tables 3 and 4). The pCAMBIA1300-35S:HPS1 plasmid was then constructed using in vitro recombination (Table 6) and transformed into wild-type Kitaake plants using Agrobacterium tumefaciens to obtain multiple transgenic lines. Total RNA from rice was then extracted using the Trizol method. The specific experimental steps are as follows: A mortar was cleaned and air-dried, then filled with alcohol and ignited to remove RNAases from the mortar at high temperature. The collected sample was frozen in liquid nitrogen, ground, and placed in a tube. An appropriate amount of Trizol reagent was added (generally, 1 mL of Trizol was added per 100 mg of sample; all data below are based on 100 mg of sample). The Trizol solution and sample powder were thoroughly mixed and allowed to stand at room temperature for 5 minutes. Add 0.2mL chloroform, vortex vigorously for 15 seconds, and let it stand at room temperature for 3 minutes; place the sample in a centrifuge, centrifuge at 12000r / min, 4℃ for 15 minutes, and carefully transfer the upper aqueous phase to a new EP tube (about 0.4mL); add an equal volume of chloroform, vortex vigorously for 15 seconds, centrifuge at 12000r / min, 4℃ for 15 minutes, and transfer the upper aqueous phase to a new EP tube (about 0.2mL) again; add the same volume of isopropanol, let it stand at room temperature for 10 minutes, centrifuge at 12000r / min, 4℃ for 10 minutes; remove the supernatant, add 0.75mL 70% ethanol, flick the bottom of the tube lightly with your hand, centrifuge at 10000r / min, 4℃ for 5 minutes, and remove the supernatant; place the EP tube in a fume hood, wait for the alcohol to evaporate, and then add 30μL RNAase-free water to fully dissolve the RNA. Reverse transcription was performed according to SuperScript from Thermo Scientific. TM III Reverse Transcriptase Kit Instructions: Synthesize the first strand of cDNA (see Tables 7 and 8). Then, use the "qHPS1" primer in Table 5 and the synthesized cDNA to prepare a quantitative PCR system (method test table 9). After the system is added, place it on the CFX96 TM PCR was performed on a Real-Time System (Bio-Rad, USA). The reaction conditions were a two-step PCR with the following program: 95°C / 30 sec; 95°C / 5 sec, 58°C / 30 sec, for a total of 39 cycles; then the temperature was increased by 0.5°C per second until it reached 95°C, thereby generating a melting curve. Quantitative analysis was performed using a CFX96 TM The Real-Time System instrument comes with 2 -ΔΔCtThe results are shown in Figure 1D. The HPS1 gene expression levels in two HPS1 overexpressed (HPS1-OE) lines (HPS1-OE#1 and HPS1-OE#4) were significantly higher than those in the wild type.

[0053] Table 5 Primer sequences related to the construction of HPS1 gene overexpression plants

[0054] Table 6 Vector in vitro recombination reaction system

[0055] Add the components in Table 7 sequentially into a DEPC-treated PCR tube.

[0056] Table 7 Reverse transcription reaction system-1

[0057] After gentle mixing, centrifugation was performed, heating was performed at 65°C for 5 min, and the mixture was placed on ice for 3 min before adding the ingredients listed in Table 8.

[0058] Table 8 Reverse transcription reaction system-2

[0059] React at 50°C for 60 min; terminate the reaction at 70°C for 15 min and store at -20°C.

[0060] Table 9 Quantitative PCR reaction system

[0061] To determine whether HPS1 protein levels were altered in HPS1-overexpressing plants, the inventors performed immunoprecipitation (IP) combined with immunoblotting of HPS1 protein in wild-type Kitaake plants and HPS1-overexpressing plants (HPS1-OE#1 and HPS1-OE#4). The specific steps were as follows: leaves were frozen and ground into a powder. A pre-chilled 50 mL low-speed centrifuge tube was removed, weighed, and the tube peeled. The powder was then added to the tube and weighed. IP buffer (50 mM HEPES [pH 7.5], 150 mM KCl, 1 mM EDTA, 0.5% Trition-X 100, 1 mM DTT, and 1 mM protease inhibitor cocktail) was added at a rate of 1 g / 2 mL and the mixture was allowed to stand at room temperature until the ice completely melted. Place the centrifuge tube on ice for 30 minutes and shake it every 10 minutes. During the incubation period, take another 1.5mL centrifuge tube, add 1mL of IP buffer and 50μL of anti-GFP magnetic beads, wash once, place on a magnetic stand, wash off the supernatant, add 0.5mL of IP buffer and place on ice. Pipette the incubated liquid into a 1.5mL centrifuge tube and centrifuge at 12300rpm, 4℃, 40min, and aspirate the supernatant into a 10mL centrifuge tube. Take 60μL of the supernatant as the test sample, then adjust the supernatant of each centrifuge tube to the same volume, resuspend the previous anti-GFP magnetic beads with 200μL of IP buffer and mix well; add 200μL of GFP magnetic beads to each centrifuge tube and rotate vertically at 4℃ for at least 4h. Add the incubated liquid to the 1.5mL centrifuge tube on the magnetic stand and aspirate the supernatant until the liquid is added. Wash cells 7-8 times with IP buffer, then add 40 μL of IP buffer for IP. Add loading buffer, heat anti-GFP magnetic beads, and analyze by immunoblotting. As shown in Figure 1, E, higher levels of HPS1 protein were enriched in the HPS1-OE#1 and HPS1-OE#4 lines compared to wild-type Kitaake plants. These results demonstrate significant HPS1 protein accumulation in two independent HPS1 overexpression lines.

[0062] Example 3 Identification of the Effect of HPS1 Gene on Regulating Rice Blast Resistance

[0063] To test whether the HPS1 gene regulates rice resistance to rice blast, the inventors first punctured leaves of three-week-old wild-type Kitaake, HPS1-KO (HPS1-KO#1 and HPS1-KO#6), and HPS1-OE (HPS1-OE#1 and HPS1-OE#4) rice seedlings and inoculated them with 5 μL of a spore suspension (5×10 5 Spores / mL) were added and the lesion size was counted after incubation at 28°C for 7 days. The results of the stab inoculation are shown in Figure 2A. Compared with wild-type Kitaake, the lesions on the leaves of HPS1-KO plants were larger, while the lesions on the leaves of HPS1-OE plants were smaller.

[0064] In the spray inoculation test, the inventors sprayed a suspension of rice blast fungus spores (5×10 5 spores / mL), and the number of lesions on each infected leaf was counted in the field after 7 days. The experimental results, shown in Figure 2B, show that compared with wild-type Kitaake, the lesions on the leaves of HPS1-KO plants were larger, while those on the leaves of HPS1-OE plants were smaller. Therefore, these results indicate that the HPS1 gene can positively regulate rice resistance to rice blast.

[0065] Example 4 Identification of the Effect of HPS1 Gene on Regulating Rice Resistance to Sheath Blight

[0066] In order to detect whether the HPS1 gene regulates rice resistance to sheath blight, the inventors placed uniformly sized mycelial blocks of the physiological subspecies AG-1-IA strain of sheath blight on the rice leaves of Kitaake, HPS1-KO (HPS1-KO#1 and HPS1-KO#6) and HPS1-OE (HPS1-OE#1 and HPS1-OE#4) plants in the tillering stage (cultured on PDA medium for 2 days), and counted the size of the lesions after incubation at 28°C for 2 days. The experimental results are shown in Figure 3. Compared with wild-type Kitaake, the lesions on the leaves of HPS1-KO plants are larger, while the lesions on the leaves of HPS1-OE plants are smaller. Therefore, the above results show that the HPS1 gene can positively regulate rice resistance to sheath blight.

[0067] Example 5 Identification of the Effect of HPS1 Gene on Regulating Rice Resistance to Bacterial Blight

[0068] To test whether HPS1 regulates rice resistance to bacterial blight, the inventors cut rice leaves from Kitaake, HPS1-KO (HPS1-KO#1 and HPS1-KO#6), and HPS1-OE (HPS1-OE#1 and HPS1-OE#4) plants at the tillering stage. The leaves were cut approximately 1 cm from the tip of the leaves. The scissors used in the experiment were pre-soaked in the physiological subspecies PXO99A (OD 600 = 0.6) (cultured on PDA medium for 2 days) suspension. Therefore, the cut leaf wounds were infected with bacterial blight and diseased. Lesion size was then counted after disease was fully developed. The experimental results are shown in Figure 4. Compared with wild-type Kitaake, lesions on the leaves of HPS1-KO plants were larger, while those on the leaves of HPS1-OE plants were smaller. Therefore, these results indicate that the HPS1 gene can positively regulate rice resistance to bacterial blight.

[0069] Example 6 Identification of the Effect of HPS1 Gene on Regulating Rice Resistance to Insect Pests

[0070] Brown planthoppers are the pests that cause the most damage to rice in actual production applications. Therefore, to test whether the HPS1 gene regulates rice resistance to pests, the inventors conducted brown planthopper infestation experiments on HPS1-related transgenic plants.

[0071] The inventors sowed approximately 15-20 seeds from two-leaf-stage Kitaake, HPS1-KO (HPS1-KO#1 and HPS1-KO#6), and HPS1-OE (HPS1-OE#1 and HPS1-OE#4) plants in a plastic cup (approximately 10 cm in diameter and 20 cm in height). The cups were then incubated with cotton soaked in Yoshida plant nutrient solution (Coolaber, Cat. No. NSP1040). When the seedlings reached the second leaf stage, the inventors introduced third-instar brown planthopper nymphs into the cups and allowed them to freely feed on the seedlings, at a density of 10 insects per seedling. When all susceptible plants died (score of 9), each seedling of other varieties or lines was scored as 0, 1, 3, 5, 7 or 9 according to the degree of damage (the main reference for this standard is J.Guo, et al., A tripartite rheostat controls self-regulated host plant resistance to insects, Nature, 618(7966):799-807, 2023). The investigation was conducted. The experimental results are shown in Figure 5. Compared with wild-type Kitaake plants, the degree of damage on the leaves of HPS1-KO plants was higher, while the degree of damage on the leaves of HPS1-OE plants was lower. This shows that the HPS1 gene can positively regulate rice resistance to insect pests represented by brown planthoppers.

[0072] In summary, the inventors discovered that overexpression of the HPS1 gene can enhance rice resistance to various fungal and bacterial diseases, such as rice blast, sheath blight, and bacterial leaf blight, as well as to insect pests caused by the brown planthopper. Knockout of the HPS1 gene, however, weakened rice resistance to these diseases and pests, among other biotic stresses. This suggests that the HPS1 gene described in this invention provides an important theoretical basis for breeding crop varieties with improved biotic stress resistance.

[0073] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of the HPS1 gene or the protein encoded by the HPS1 gene in regulating plant disease resistance, characterized in that: The nucleotide sequence of the HPS1 gene is shown in SEQ ID NO.1; the amino acid sequence of the protein is shown in SEQ ID NO.2; The regulation includes knocking out the HPS1 gene to reduce the disease resistance of the plant and overexpressing the HPS1 gene to increase the disease resistance of the plant; The disease resistance includes the ability to resist diseases caused by fungi and the ability to resist diseases caused by bacteria; The fungus is a pathogen that causes rice blast and / or a pathogen that causes sheath blight; the bacteria is a pathogen that causes bacterial blight; The plant is rice.

2. Use of the HPS1 gene or the protein encoded by the HPS1 gene in regulating the insect pest resistance of plants, characterized in that: The nucleotide sequence of the HPS1 gene is shown in SEQ ID NO.1; the amino acid sequence of the protein is shown in SEQ ID NO.2; The regulation includes knocking out the HPS1 gene to reduce the insect pest resistance of the plant and overexpressing the HPS1 gene to increase the insect pest resistance of the plant; The insect pest is caused by brown planthopper; and the plant is rice.

Images