Application of gene mos4 and its encoded protein in regulating plant resistance to phytopathogenic pythium

Abstract

The application belongs to the technical field of bio-agriculture, and particularly relates to a gene MOS4 and application of an encoded protein thereof in regulation of plant resistance to parasitic Phytophthora. MOS4 Through gene function research, it is found that overexpression of the gene MOS4 significantly improves the plant resistance to parasitic Phytophthora, and that the expression of the gene MOS4 weakens the plant resistance to parasitic Phytophthora, and that the gene MOS4 positively regulates the plant resistance to parasitic Phytophthora. Further immune mechanism research finds that the gene MOS4 regulates the related genes of a salicylic acid (SA) signal path, interacts with a transcription factor ERF019, and jointly activates the plant immune response. In addition, a dual luciferase experiment proves that the gene MOS4 and the gene ERF019 cooperatively regulate the transcription of PR1. The application provides a new molecular target for prevention and treatment of plant diseases caused by parasitic Phytophthora, and has important agricultural application value.

Description

Technical Field

[0001] This invention belongs to the field of bio-agricultural technology, specifically relating to genes. MOS4 The application of its encoded proteins in regulating plant resistance to Phytophthora parasiticis. Background Technology

[0002] Phytophthora parasiticum ( Phytophthora parasitica *Phytophthora parasitica* is a heterothallic, semi-vitroic pathogen. During its asexual reproduction stage, it produces asexual sporangia, biflagellate zoospores, and chlamydospores. The zoospores produced from the sporangia rest on the surface of the infected host and then germinate. The germinated germ tubes form appressoriums at their tips, which then invade the plant roots and leaves. The invasive hyphae develop into haustoria, and under suitable conditions, produce numerous sporangia on the surface of the infected plant, rapidly infecting it. *Phytophthora parasitica* has a wide host range and causes serious damage to agricultural production and natural ecosystems.

[0003] Currently, the main means of controlling diseases caused by Phytophthora parasiticus is still fungicides. However, excessive or improper use of fungicides can easily lead to drug resistance, pesticide residues, and environmental pollution. Breeding Phytophthora parasiticus-resistant varieties has become an effective means of solving the existing problems with fungicides. However, Phytophthora parasiticus exhibits high genetic variability, and its dynamic genome includes a large number of effector proteins. These regions may tolerate more non-allelic recombination and other forms of structural variation, promoting increased virulence of Phytophthora parasiticus and other changes favorable to the pathogen. Therefore, finding more Phytophthora parasiticus-resistant genes and studying their immune mechanisms in the process of interaction with plants is of great significance for solving the current technical problems in the control of diseases caused by Phytophthora parasiticus.

[0004] MOS4 (Modifier of snc1,4) encodes a protein homologous to human breast cancer amplified sequence 2 (BCAS2) and is an important member of the MAC complex. While snc1 mutants have been reported to exhibit resistance to *Pseudomonas syringae* ES4326 and *Noco2* in plants, no reports have yet been found regarding MOS4's association with resistance to *Phytophthora parasitica*. The effector proteins of *Pseudomonas syringae*, *Persona*, and *Phytophthora parasitica* differ. Therefore, investigating whether MOS4 participates in the regulation of plant resistance to *Phytophthora parasitica* and its mechanism of action is of great significance for the control of diseases caused by *Phytophthora parasitica*. Summary of the Invention

[0005] Based on the above-mentioned technical problems, the present invention screens and obtains genes. MOS4 The CDS fragment of this gene was obtained through genetic engineering. Research was conducted on the disease resistance and related immune molecules of mutant plants and plants overexpressing this gene after infection of leaves and roots with Phytophthora parasiticus. The study revealed that the gene... MOS4This invention plays a positive regulatory role in plant resistance to Phytophthora parasiticum, and provides new gene resources for the prevention and control of Phytophthora parasiticum from a molecular biology perspective.

[0006] On the one hand, this invention provides genes MOS4 Application in regulating plant resistance to Phytophthora parasitoids, by overexpressing the gene. MOS4 The gene enhances plant resistance to Phytophthora parasiticum. MOS4 The nucleotide sequence of the CDS region is shown in SEQ ID NO:1.

[0007] Furthermore, in the aforementioned application, the gene MOS4 Through interaction with transcription factors ERF019 The interaction regulates plant resistance to Phytophthora parasiticum, and the genes mentioned MOS4 With transcription factors ERF019 Joint regulation PR1 Transcription.

[0008] Secondly, this invention also provides the application of protein MOS4 in regulating plant resistance to Phytophthora parasiticus, increasing the expression level or enhancing the activity of protein MOS4 to improve plant resistance to Phytophthora parasiticus; wherein protein MOS4 is derived from a gene. MOS4 The amino acid sequence of the protein MOS4 is as shown in SEQ ID NO:2.

[0009] Thirdly, the present invention also provides a method for reducing plant resistance to Phytophthora parasiticus, wherein the method reduces gene resistance within the plant. MOS4 The expression of this gene reduces plant resistance to Phytophthora parasiticum. MOS4 The nucleotide sequence of the CDS region is shown in SEQ ID NO:1.

[0010] Fourthly, a method to enhance plant resistance to Phytophthora parasiticum involves overexpressing genes within the plant. MOS4 The gene enhances plant resistance to Phytophthora parasiticum. MOS4 The nucleotide sequence of the CDS region is shown in SEQ ID NO:1.

[0011] Furthermore, in the method, by constructing a structure containing genes MOS4 The overexpression vector, via Agrobacterium-mediated transformation, expresses the gene-containing... MOS4 The overexpression vector was transformed into the plant to overexpress the gene. MO S4, the gene MOS4 The nucleotide sequence of the CDS region is shown in SEQ ID NO:1.

[0012] Fifthly, the present invention also provides a method for cultivating plant varieties resistant to Phytophthora parasitica, comprising introducing genes into the plant. MOS4 Constructing genesMOS4 Overexpressing plants, the gene MOS4 The nucleotide sequence of the CDS region is shown in SEQ ID NO:1.

[0013] Furthermore, in the method, the gene MOS4 In overexpressing plants, when Phytophthora parasiticum invades, the plants enhance their resistance to Phytophthora parasiticum by regulating the expression of SA-related genes.

[0014] Furthermore, in the method, the SA-related gene is an SA signaling pathway marker gene. PR1 Genes related to SA synthesis ICS1 .

[0015] Furthermore, in the method, the plant is Arabidopsis thaliana.

[0016] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0017] This invention discovers genes MOS4 The positive regulatory role of genes in plant resistance to Phytophthora parasiticus provides new genetic resources for disease-resistant breeding; gene discovery MOS4 By upregulating the SA synthesis gene ICS1 and SA signaling marker genes PR1 The expression of this substance enhances the plant's immune response, providing new molecular evidence for disease resistance mechanisms.

[0018] This invention demonstrates, through Co-IP and dual-luciferase experiments, that genes MOS4 With transcription factors ERF019 Interacting and jointly regulating PR1 Transcription provides a new regulatory node for the plant immune regulatory network.

[0019] This invention utilizes gene overexpression MOS4 This significantly improves plant disease resistance and provides a new genetic improvement approach for cultivating Phytophthora resistant crops.

[0020] In summary, this invention reveals the gene MOS4 The study elucidates the key role of Phytophthora in plant resistance and the mechanism by which it regulates plant immunity through the interaction of the SA signaling pathway and ERF019, providing new theoretical basis and technical means for disease-resistant breeding and possessing significant scientific and applied value. Attached Figure Description

[0021] Figure 1 For genes MOS4 The graph shows the test results of resistance to *Phytophthora parasiticus* infection by T-DNA insertion mutants. (a) represents... mos4 mutant mos4a , mos4b existMOS4 Schematic diagram of T-DNA insertion sites; b is MOS4 T-DNA insertion mutant mos4a , mos4b Image 1: Observation of Phytophthora zoospore lesions on leaves of wild-type Col-0; c: Phytophthora parasiticum in mutants. mos4a , mos4b d is a statistical result of the infection degree on leaves of wild-type Col-0; d is a statistical result of the biomass of Phytophthora parasiticus on infected leaves by real-time quantitative PCR analysis; e is an observation of the mycelial infection of Phytophthora parasiticus in Arabidopsis roots; f is... MOS4 T-DNA insertion mutant mos4a , mos4b Statistical results of infection length of roots inoculated with Phytophthora infestans 24 hours after inoculation with wild-type Col-0; g represents T-DNA insertion mutant. mos4a , mos4b middle MOS4 The level of expression. "*" indicates... P <0.05, "**" indicates P <0.01, "***" indicates P <0.001.

[0022] Figure 2 For genes MOS4 Figure showing the results of resistance assays for Phytophthora parasiticus infection in overexpressing plants. 'a' represents the gene. MOS4 OE1 and gene expression in plants MOS4 Figure b shows the observation of lesions on leaves of OE3 and wild-type Col-0 plants inoculated with Phytophthora parasitoids zoospores; b shows the lesions caused by Phytophthora parasitoids in the gene... MOS4 OE1 gene in plants MOS4 Statistical results of infection degree on leaves of OE3 and Col-0 plants; c is the biomass of Phytophthora parasiticus on infected leaves analyzed by real-time quantitative PCR; d is the gene... MOS4 OE1 gene in plants MOS4 Figure showing the degree of infection of *Phytophthora parasitica* mycelia in the roots of OE3 and Col-0 plants; e represents the gene. MOS4 OE1 gene in plants MOS4 The mycelial infection length of *Phytophthora parasitica* 24 hours after inoculation of the roots of OE3 and Col-0 plants was measured; the results are expressed as the mean ± standard error of the infection length of 18 plants. t Significance analysis was performed; f represents the number of genes. MOS4 OE1 gene in plants MOS4 In the OE3 and Col-0 of the over-exposed plants MOS4 The level of expression. "*" indicates... P <0.05, "**" indicates P<0.01, "***" indicates P <0.001.

[0023] Figure 3 For genes MOS4 Schematic diagram of expression patterns in response to infection by Phytophthora parasitica.

[0024] Figure 4 For genes MOS4 Expression pattern detection results under SA induction. a) Distribution of the SA-responsive TCA element on the MOS4 and ERF019 promoters; b) Gene expression at different time points after SA treatment. MOS4 The level of expression. "*" indicates... P <0.05, "***" indicates P <0.001.

[0025] Figure 5 This diagram illustrates the expression patterns of SA signaling pathway marker genes and SA synthesis-related genes in the mos4 mutant. a) shows the expression of the SA signaling pathway marker gene PR1; b) shows the expression of SA synthesis-related genes. ICS1 The expression of c; c is the gene related to SA synthesis. PAL1 The expression.

[0026] Figure 6 Figure 1 shows the results of the immunoprecipitation assay of protein MOS4 and E protein RF019 in plants. Figure 2a shows the results of immunoprecipitation of protein samples using Anti-GFP magnetic beads; Figure 3b shows the results of immunoprecipitation of protein samples using Anti-FLAG magnetic beads.

[0027] Figure 7 Gene testing for Dual-LUC MOS4 With transcription factors ERF019 right PR1 The diagram shows the transcriptional regulation results. "**" indicates... P <0.01, "***" indicates P <0.001. Detailed Implementation

[0028] The technical solution of the present invention will be described below with reference to the embodiments. However, the present invention is not limited to the following embodiments.

[0029] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the present invention.

[0030] Unless otherwise specified, the experimental and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0031] Example 1

[0032] This example is about genes. MOS4 Obtaining the CDS fragment.

[0033] The Arabidopsis gene was obtained through the Ensembl Plants website (https: / / plants.ensembl.org / Triticum_aestivum / Info / Index). MOS4 The nucleotide sequence of the CDS, the Arabidopsis gene MOS4 The nucleotide sequence of the CDS is shown in SEQ ID NO:1, and the amino acid sequence of its encoded protein MOS4 is shown in SEQ ID NO:2. The gene was amplified by PCR using cDNA from wild-type Arabidopsis thaliana Col-0 as a template. MOS4 The CDS fragment was amplified by PCR using primers MOS4-F and MOS4-R. Primer sequences are shown in Table 1. The amplified gene... MOS4 CDS fragment sequencing, gene MOS4 The sequencing results of the CDS fragment are shown in SEQ ID NO:1.

[0034] SEQ ID NO:1 is as follows:

[0035] ATGGCGACGAACAATGGTGATGTCTTGATGTTGGAGGCGACGCCGGAGGCTGCGAGGCCCTGGGCTAGTGCGGCGAATGCAGAAGTTATCGATGCGCTTCCTTATATAGACGACGACTATGGCAATCCATTGATTAAGTCGGAGGTAGACCGTTTGGTGGAGGAAGAGATGCGTCGGAGCTCTAAGAAGCCAGCTGACTTTCTAAAGGACTTGCCTCCTCTTCCGAAGTTCGATTTTAAGAACTGCCCTGTTCTTGGCAAAGAGTATGAGCGTGTTAGAGCTGGGAAGCCTCCTGTGCGGATAGATTTCGAATCCCGATACAAACTTGAAATGCCACCTGCCAATAAGAGGAATGATGATGCTGCCTGGAAGCAGTATCTTCAGAAGAATCAACGGTCATTGCAACAGAAGCTGATTGAGCTTGAGAATTTGGAATTGATGTCAAAACTTGGCCCCGAGCTTTGGAGACAGAACAACCATCGCCTTGAAGTATTTTTGACCAGAATGCAAAGACTAGCACAGGAGCAGAATGAGGAAATTGAAAAAGTAAATCGGGAAAGGAAGTATCATCAGCAAACCACATCATACGAGCTCAATGCTCTATCTCAAGAATGGAGACAGCTCTGTGTTAAGAATATGGAGATTCAGTCTGCTTGTGCCATGCTTGAGACACAGATCGATTCGTTCAAGAAAGAAGCTGCTGAAAGGGGTTGGAACTTAGAAGAGAAACTAGAGAACGTCGAGCCACTTCAAATGCAATGA。

[0036] SEQ ID NO: 2 is as follows:

[0037] MATNNGDVLMLEATPEAARPWASAANAEVIDALPYIDDDYGNPLIKSEVDRLVEEEMRRSSKKPADFLKDLPPLPKFDFKNCPVLGKEYERVRAGKPPVRIDFESRYKLEMPPANKRNDDAAWKQYL QKNQRSLQQKLIELENLELMSKLGPELWRQNNHRLEVFLTRMQRLAQEQNEEIEKVNRERKYHQQTTSYELNALSQEWRQLCVKNMEIQSACAMLETQIDSFKKEAAERGWNLEEKLENVEPLQMQ.

[0038] Table 1 Primer sequence listing

[0039]

[0040] Example 2

[0041] This example demonstrates gene testing. MOS4 The effect of mutants on resistance to Phytophthora parasitica.

[0042] Gene of MOS4 T-DNA insertion mutant mos4a (SALK_019535) and mos4b (SALK_090851), purchased from the Arabidopsis Biological Resource Center (ABRC), the aforementioned mos4a and mos4b The two mutants were respectively in MOS4 Mutation sites are inserted into the first intron and the first exon. A schematic diagram of the mutation sites is shown below. Figure 1 As shown in Figure a. Real-time quantitative PCR was used for determination. mos4a and mos4b Genes in the two mutants MOS4 expression level ， Test results are as follows Figure 1 As shown in g, the results show the insertion mutant. mos4a and mos4b Zhonggen MOS4 The expression levels of all of them were downregulated.

[0043] Analysis using in vitro leaf parasitic Phytophthora inoculation test MOS4 mutant mos4a and mos4bImmunological function. Fully expanded rosette leaves from the same leaf position were taken from wild-type Col-0, mutant mos4a, and mos4b plants that had grown for 4 weeks. The leaves were placed with the underside facing upwards, and the petioles were placed between two layers of defatted cotton strips completely moistened with dH2O. Each group contained 10-12 leaves. The mid-section of both sides of the underside of the Arabidopsis leaves was scrambled, and 15 μL of *Phytophthora parasitica* zoospore suspension was inoculated at the scrambled sites. The plants were incubated at 23°C. Photographs were taken 3 days after inoculation, and the disease incidence on the leaves was observed and graded using a fluorescence microscope. Based on the disease incidence on the Arabidopsis leaves, the infection of *Phytophthora parasitica* mycelia on the leaves was observed using a fluorescence microscope and graded from 0 to 4. Level 0 was defined as no leaf infection; Level 1 was defined as the area of ​​infection on both sides of the leaf being less than half the leaf area; Level 2 was defined as the area of ​​infection on one side of the leaf being less than half the leaf area; Level 3 was defined as the area of ​​infection on both sides of the leaf being greater than half the leaf area; and Level 4 was defined as the entire leaf being infected by Phytophthora parasiticus. Further analysis of mos4a and its related pathogens was conducted by comparing the disease incidence on leaves of different plants. mos4b Disease resistance of mutants. (From...) Figure 1 From b, we can know that genes MOS4 Insertion mutant mos4a , mos4b Compared with the wild-type Col-0 leaves, the lesion area after inoculation with Phytophthora parasitica zoospores was significantly larger than that of the control group, wild-type Col-0. Figure 1 From c, we can see that the Arabidopsis gene MOS4 Insertion mutant mos4a , mos4b The disease is more severe compared to the wild-type Col-0.

[0044] Collect mutants infected with Phytophthora infestans mos4a , mos4b DNA was extracted from leaves of wild-type Col-0, and the biomass of *Phytophthora parasitica* was detected using real-time quantitative PCR. The quantitative analysis results are as follows: Figure 1 As shown in d, mos4 mutant mos4a , mos4b The colonization rate of *Phytophthora parasiticus* on infected leaves was significantly higher than that of the wild-type Col-0. This indicates that the gene... MOS4 Insertion mutant mos4a and mos4b It is more susceptible to Phytophthora parasiticum infection compared to the wild type of Arabidopsis thaliana (Col-0).

[0045] Arabidopsis mos4 mutant mos4a , mos4b Inoculation tests were conducted on root parasitic Phytophthora mycelial blocks in petri dishes.

[0046] Arabidopsis seeds were sown on 1 / 2 MS+S medium and incubated vertically at 23°C. One week later, the Arabidopsis seedlings were gently transferred to 1 / 2 MS-S medium. *Phytophthora parasiticus* Pp016 was cultured on 5% carrot solid medium plates at 23°C for 4 days. Using a yellow pipette tip (approximately 5 mm inner diameter), circular mycelial cakes were formed along the edge of fresh *Phytophthora parasiticus* hyphae. The mycelial-containing side was then placed on the middle of the root of an Arabidopsis root on sucrose-free 1 / 2 MS (1 / 2 MS-S) medium and incubated vertically at 23°C. After 24 hours, the length of *Phytophthora parasiticus* infection on the root was observed and marked using a fluorescence microscope. The length of infected Arabidopsis roots was measured and statistically analyzed. Results are as follows: Figure 1 As shown in e and f, compared with wild-type Col-0, the mutant mos4a , mos4b The infected length of the root is longer. mos4 mutant mos4a , mos4b The roots showed greater susceptibility to Phytophthora parasiticum infection, consistent with the resistant phenotype observed in the leaves. In summary, the results of this example indicate that the gene... MOS4 It may be regulating the infection of Arabidopsis thaliana by Phytophthora.

[0047] Example 3

[0048] This example is for testing genes. MOS4 The effect of overexpression on the resistance of plants to Phytophthora parasitica.

[0049] Using Col-0 cDNA as a template for PCR amplification, the gene was amplified. MOS4 CDS fragments. Genes MOS4 The CDS fragment was cloned into the linearized backbone pART27-pKANNIBAL, which was double-digested with restriction endonucleases XhoI / XbaI, to obtain the recombinant plasmid pART27-pKANNIBAL- MOS4 Used to build genes MOS4 Overexpressing plants.

[0050] Following the Agrobacterium electroporation competent transformation method, the recombinant plasmid pART27-pKANNIBAL- MOS4 Transfect the plasmid into GV3101 competent cells. Single colonies of the recombinant plasmid grown on antibiotic-resistant plates containing rifampin, gentamicin, and spectinomycin were picked and gently shaken in LB broth until orange-yellow. After centrifugation at 4000 g for 5 min, the resuspended cells were gently resuspended in MES broth containing acetylsuccinone, and the OD of the resuspended solution was adjusted. 600 A concentration of 0.3-0.4 g / L was used for leaf injection in Arabidopsis thaliana after incubation at room temperature for 1-4 hours. Two genes driven by strong 35S promoters were obtained using an Agrobacterium-mediated inflorescence immersion method. MOS4Overexpression of stable transformation materials OE1 and OE3 (genes) MOS4 Overexpression of plant OE1 and gene MOS4 Overexpressing plants (OE3) were used to determine the gene expression using real-time quantitative PCR. MOS4 Overexpression of plant OE1 and gene MOS4 Genes in overexpressed plants OE3 MOS4 Expression level, results as follows Figure 2 As shown in f, genes MOS4 Overexpression of plant OE1 and gene MOS4 Genes in overexpressed plants OE3 MOS4 The expression level was adjusted upward.

[0051] On genes MOS4 Overexpression of plant OE1 and gene MOS4 In vitro leaves of plants overexpressing OE3 and wild-type Col-0 were inoculated with zoospores of *Phytophthora parasitica*, following the same experimental procedure as in Example 2. The results of inoculation of detached leaves are as follows: Figure 2 As shown in Figures a and b, compared to wild-type Col-0, the gene... MOS4 Overexpression of plant OE1 and gene MOS4 Overexpression of this substance resulted in leaves that were more resistant to Phytophthora parasiticum infection.

[0052] Extracting genes from Phytophthora parasiticum infection MOS4 Overexpression of plant OE1 and gene MOS4 DNA was overexpressed in leaf samples from OE3 and wild-type Col-0 plants, and the biomass of *Phytophthora parasitica* in the leaves was detected by real-time quantitative PCR. The quantitative results are as follows: Figure 2 As shown in c, the gene MOS4 Overexpression of plant OE1 and gene MOS4 The colonization rate of Phytophthora parasiticus on leaves infected with OE3 from overexpressing plants was significantly lower than that of wild-type Col-0.

[0053] On genes MOS4 Overexpression of plant OE1 and gene MOS4 Overexpressing OE3 plants were inoculated with Phytophthora mycelium in a dish, following the same procedure as in Example 2. Results are as follows: Figure 2 As shown in d and e, the results show that compared with wild-type Col-0, the gene... MOS4 Overexpression of plant OE1 and gene MOS4 Overexpressing OE3 resulted in shorter root lengths when infected by Phytophthora parasiticus hyphae, indicating greater resistance to Phytophthora parasiticus infection. This is related to the gene... MOS4 Overexpression of plant OE1 and gene MOS4 Overexpressing OE3 plants exhibited consistent resistance phenotypes to Phytophthora parasiticus in their leaves. These experimental results indicate that, compared to wild-type Col-0, the gene... MOS4 Overexpression of plant OE1 and geneMOS4 Plants overexpressing OE3 showed greater resistance to Phytophthora parasiticus infection in both roots and leaves. In summary, the gene... MOS4 Positive regulation of Arabidopsis resistance to Phytophthora can be applied to the regulation of plant resistance to Phytophthora and to the breeding of Phytophthora-resistant and disease-resistant varieties.

[0054] Example 4

[0055] This example is about genes. MO An investigation into the immune mechanism by which S4 positively regulates plant resistance to Phytophthora parasiticus.

[0056] 4.1 Analysis of the expression pattern of gene MOS4 in response to infection by Phytophthora parasitica:

[0057] Col-0 leaf samples infected with Phytophthora parasiticus at 0, 3, 6, 12, 24, and 48 hours were selected. RNA was extracted and detected using real-time quantitative PCR technology. MOS4 The expression level. Real-time quantitative PCR results are as follows: Figure 3 As shown, after inoculation with Phytophthora parasitica, the gene... MOS4 The expression level of the gene was downregulated from 0 to 3 hours, and then induced to be upregulated from 3 to 48 hours, indicating that the gene... MOS4 It can respond to parasitic fungal infections.

[0058] 4.2 Expression pattern analysis of gene MOS4 under SA induction:

[0059] Gene analysis MOS4 and ERF019 Cis-acting elements on the promoter sequence. The results are as follows: Figure 4 As shown in Figure a, it displays MOS4 Both the promoter sequences of *Arabidopsis thaliana* and *ERF019* contain the salicylic acid (SA) response element TCA. Therefore, RT-qPCR was used to detect the gene expression in *Arabidopsis thaliana* Col-0 leaves at different time points after SA treatment. MOS4 Expression levels, assess gene expression levels MOS4 Whether the expression level is induced by salicylic acid (SA) treatment. The detection results are as follows: Figure 4 As shown in b, the gene is displayed. MOS4 It can be induced by SA, and its expression is downregulated by SA.

[0060] 4.3 Testing the expression of PR1, a marker gene of the SA signaling pathway, and ICS1, a gene related to SA synthesis, in the mos4 mutant:

[0061] Detection of parasitic fungal infection genes mos4 mutant mos4a , mos4bGenes related to SA synthesis in wild-type Col-0 plants at 0h, 6h, and 24h time points. ICS1 , PAL1 and SA signaling pathway marker genes PR1 In genes mos4 mutant mos4a , mos4b The expression levels in wild-type Col-0 and [other samples were observed]. Results are as follows: Figure 5 As shown, Figure 5 Images a and b show marker genes for the SA signaling pathway. PR1 Genes related to SA synthesis ICS1 In genes mos4 mutant mos4a and mos4b The expression levels of Phytophthora parasitica at 0, 6, and 24 hours after inoculation were significantly lower than those of wild-type Col-0. This indicates that the gene... MOS4 It may participate in plant resistance to Phytophthora parasitica by affecting the expression of genes related to the SA signaling pathway.

[0062] 4.4 Determination of the interaction between proteins MOS4 and ERF019 in plants:

[0063] The interaction between proteins MOS4 and ERF019 in plants was detected using co-immunoprecipitation (Co-IP) and bimolecular complementary fluorescence (BiFC) assays. In the Co-immunoprecipitation experiment, ERF019-GFP and FLAG-MOS4 were co-injected into leaves of *Nicotiana benthamiana* (Tobacco Bengal) in the experimental group, and GFP and FLAG-MOS4 were co-injected into the control group. Total protein was extracted from the leaves after three days of expression and immunoprecipitated using magnetic agarose beads (Anti-GFP magnetic beads and Anti-FLAG magnetic beads) that bound GFP and FLAG, respectively. The results are shown below. Figure 6 As shown, by Figure 6 As shown in section a, using Western blotting to detect proteins enriched by Anti-GFP magnetic beads, ERF019-GFP can enrich FLAG-MOS4, while GFP cannot enrich FLAG-MOS4; from Figure 6 As shown in Figure b, Western blotting analysis revealed that proteins enriched by Anti-FLAG magnetic beads, specifically FLAG-MOS4, were able to enrich ERF019-GFP. This indicates that proteins ERF019 and MOS4 can interact in plant cells, suggesting that MOS4 may regulate plant resistance to Phytophthora parasiticum infection through its interaction with ERF019.

[0064] 4.5 Test genes MOS4 With transcription factors ERF019 right PR1 Transcriptional regulation:

[0065] The recombinant vectors pGreenII 62SK-MOS4, pGreenII 62SK-ERF019, and pGreenII 0800-luc-ProPR1 were co-expressed on tobacco leaves, and dual-luciferase activity was detected. The results are as follows: Figure 7 As shown in the figure, the experiment showed that, compared with the negative control group, a total of 62SK empty vector and PR1 Compared to the promoter, it co-expresses MOS4, ERF019 and PR1 The promoter-recombinant vector had a higher LUC / REN ratio. However, co-expression of ERF019 and... PR1 The ratio of promoter-recombinant vectors was lower than that of the control group, indicating that the gene... MOS4 Affecting ERF019 PR1 Transcriptional regulation.

[0066] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. Overexpressed genes MOS4 In improving the resistance of Arabidopsis thaliana to Phytophthora ( Phytophthora parasitica Its application in sex is characterized by, The gene MOS4 The nucleotide sequence of the CDS region is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The gene MOS4 Through interaction with transcription factors ERF019 The interaction regulates the resistance of Arabidopsis thaliana to Phytophthora, the gene MOS4 With transcription factors ERF019 Joint regulation PR1 Transcription.

3. A method to enhance the resistance of Arabidopsis thaliana to Phytophthora ( Phytophthora parasitica The method of sex is characterized by, Gene overexpression in Arabidopsis thaliana MOS4 Enhance Arabidopsis thaliana's resistance to Phytophthora parasiticum ( Phytophthora parasitica ) sex, the gene MOS4 The nucleotide sequence of the CDS region is shown in SEQ ID NO:

1.

4. The method according to claim 3, characterized in that, By constructing genes MOS4 The overexpression vector, via Agrobacterium-mediated transformation, expresses the gene-containing... MOS4 The overexpression vector was transformed into Arabidopsis thaliana to overexpress the gene. MOS4 .

5. A method for cultivating parasitic resistant Phytophthora ( Phytophthora parasitica The method for cultivating Arabidopsis thaliana varieties is characterized by, Including the introduction of genes into Arabidopsis thaliana MOS4 Constructing genes MOS4 Overexpressing plants, the gene MOS4 The nucleotide sequence of the CDS region is shown in SEQ ID NO:

1.

6. The method according to claim 5, characterized in that, The gene MOS4 In overexpressing plants, when *Phytophthora infestans* invades, *Arabidopsis thaliana* enhances its resistance to *Phytophthora infestans* by regulating the expression of SA-related genes, which are marker genes of the SA signaling pathway. PR1 Genes related to SA synthesis ICS1 .

Images