Rice receptor-like kinase OsLTK1 and application thereof in resistance of magnaporthe oryzae

Abstract

The invention belongs to the technical field of biology, and discloses a rice receptor-like kinase OsLTK1 and application thereof in resistance of magnaporthe oryzae. It is found for the first time that receptor-like kinase OsLTK1 positively regulates the resistance of rice to magnaporthe oryzae. The research shows that the transgenic plant of the OsLTK1 gene overexpressed in the rice has obvious resistance to the magnaporthe oryzae. Therefore, the OsLTK1 gene can be used for cultivating rice blast resistant rice varieties, and has great application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a rice receptor kinase OsLTK1 and an application thereof in resistance to rice blast fungus. Background Art

[0002] Rice is a major food crop in my country, with a cultivated area accounting for approximately 1 / 4 of the country's total grain-planted area. However, rice diseases caused by the rice blast fungus (Magnaporthe oryzae) have seriously affected the normal production of rice, reducing the quality and commercial value of rice. Rice blast, along with rice sheath blight, rice false smut, and rice virus diseases, are known as the four major rice diseases. Rice blast occurs in both northern and southern rice-growing areas of my country, causing a reduction in rice production in my country. Currently, the prevention and control of rice blast fungus mainly focuses on the development of new disease-resistant rice varieties, supplemented by cultivation measures and chemical control. However, rice blast fungus is very prone to mutation, resulting in disease-resistant rice varieties often losing their resistance to rice blast fungus within a few years of their introduction. In addition, chemical control methods are too single and often cannot fundamentally solve the problem. Extensive use also causes environmental pollution. Therefore, a deep understanding of the molecular mechanism of the interaction between rice and rice blast fungus will not only help to develop and apply broad-spectrum and effective control strategies against rice blast fungus, but also provide a theoretical basis for exploring new genetic resources for breeding rice blast fungus resistance, which has important application value for the sustainable production of rice in the future.

[0003] Plants have evolved two immune mechanisms to resist pathogen infection. The first is the pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), and the second is the effector-triggered immunity (ETI). When plants are attacked by external pathogens, specific receptors (PRRs) on the surface of plants S ) recognizes PAMPs on the surface of pathogens SAt this time, the plant's PTI immune mechanism will be activated first, inducing the plant to produce a series of immune responses to hinder the invasion of pathogens. In the long-term process of co-evolution with plants, pathogens have acquired the ability to secrete effector proteins to interfere with the plant's PTI immune mechanism. To further limit the infection of pathogens, disease resistance-related proteins in plant cells can specifically recognize certain effector factors and trigger the plant's second immune mechanism (ETI) [BigeardI J, Colcombet J, Hirt H. Signaling mechanisms in pattern-triggered immunity (PTI) [J]. Mol Plant, 2015, 8(4): 521-39.;Jones JD, DanglJ L. The plant immune system [J]. Nature, 2006, 444(7117): 323-9.;Weigel D,Dangl J L. 10 years of Current Opinion in Plant Biology 1997-2007 [J]. CurrOpin Plant Biol, 2007, 10(6): 543-5.]. Plant PTI and ETI immune mechanisms activate disease resistance factors, produce reactive oxygen species, and induce callose deposition in the process of restricting infection by external pathogens. Reactive oxygen species (ROS) can directly kill invading pathogens or act as signaling molecules to activate downstream immune responses, enabling plants to gain stronger pathogen resistance. Studies have found that plant ETI immune mechanisms can enhance the expression of the NADPH oxidase RBOHD, while cytoplasmic receptor kinases in the PTI immune mechanism can promote RBOHD activation [Yuan M, Jiang Z, Bi G, et al. Pattern-recognition receptors are required for NLR-mediated plant immunity[J]. Nature, 2021, 592(7852): 105-9.]. In addition, PTI triggered by PAMPs is stable, persistent, and broad-spectrum, and understanding its mechanism has important theoretical and practical value for improving durable disease resistance in crops. Plant receptor-like kinase (RLK) is a type of pattern recognition receptor (PRRs) on the surface of plants. It is one of the largest transmembrane signaling protein families in plants. After sensing external signals in the extracellular domain, it transmits signals through the intracellular kinase domain to regulate plant growth and development, immune defense, and stress response.The typical structure of RLK includes an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular kinase. Based on the characteristics of the extracellular domain, RLK is divided into more than 20 subfamilies, such as LRR-RLK and Lys-RLK. More than 600 members have been identified in Arabidopsis [Shiu SH, Bleecker A B. Plant receptor-like kinase gene family: diversity, function, and signaling [J]. Science's STKE: signal transduction knowledge environment, 2001, 2001(113): re22.]. In recent years, some studies have found that RLK proteins play a very important role in the process of rice resistance to rice blast infection. At present, 17 RLK proteins have been identified in rice to participate in resistance to rice blast fungus. For example, OsBDR1 negatively regulates rice resistance to rice blast fungus by inhibiting the jasmonate signaling pathway and terpenoid biosynthesis pathway by interacting with mitogen-activated protein kinase 3 (MPK3) [Wang L,Xu G, Li L, et al. The OsBDR1-MPK3 module negatively regulates blast resistance by suppressing the jasmonate signaling and terpenoid biosynthesispathway [J]. Proc Natl Acad Sci USA, 2023, 120(13): e2211102120.]. In addition, salicylic acid (SA) and jasmonic acid (JA) significantly accumulated in ALS-overexpressing rice, significantly enhancing its resistance to rice blast fungus. In terms of ROS regulation, the SDS2 protein interacts with OsRLCK118 and OsRLCK176 to phosphorylate the NADPH oxidase OsRbohB, stimulating ROS production and actively regulating immune responses [Fan J, Bai P, Ning Y, et al. The Monocot-Specific Receptor-like Kinase SDS2 Controls Cell Death and Immunityin Rice [J]. 2018: 498-510.e5.]. OsRLCK118 enhances rice resistance to the rice blast fungus by affecting ROS levels and defense hormone-related signaling pathways, while silencing it increases rice susceptibility to the fungus. RLK proteins also play a crucial role in rice growth and development.For example, the PWL1 gene encodes a lectin receptor-like protein kinase (LecRLK), which is essential for maintaining chlorophyll content in rice leaves. Loss of PWL1 function leads to decreased chlorophyll levels and early senescence [Xu J, Wang C, Wang F, et al. PWL1, a G-type lectinreceptor-like kinase, positively regulates leaf senescence and heat tolerancebut negatively regulates resistance to Xanthomonas oryzae in rice [J]. Plantbiotechnology journal, 2023, 21(12): 2525-45.]. The brassinosteroid (BR) receptor OsBRI1 can regulate rice plant height and tiller number through the BR signaling pathway. After BR binds to the extracellular domain of the OsBRI1 protein, the kinase domain of OsBRI1 is activated, phosphorylating the downstream target OsBSK1, inhibiting the phosphatase BSU1, and ultimately relieving the inhibitory effect of BIN2 on BZR1, promoting the expression of tillering-related genes [Zhao J, Wu C, Yuan S, et al. Kinase activity of OsBRI1is essential for brassinosteroids to regulate rice growth and development[J]. Plant science: an international journal of experimental plant biology,2013, 199-200: 113-20.].

[0004] Crop breeders hope to create new high-yield and disease-resistant rice varieties. Identifying genes that precisely regulate rice growth and disease resistance at specific growth stages or tissues holds promise for addressing this challenge. Rapid advances in genome sequencing and bioinformatics analysis have enabled the identification of more RLK genes in rice, paving the way for further research into the biological functions and molecular mechanisms of RLK proteins, and facilitating the development of new disease-resistant, high-yield rice varieties through molecular breeding. Summary of the Invention

[0005] The present invention has been discovered through research that transgenic rice overexpressing the OsLTK1 gene exhibits significant resistance to the rice blast fungus. After the OsLTK1 gene is knocked out, the rice exhibits a susceptible phenotype, while the growth of the knockout rice material is not affected. This led to the present invention.

[0006] The present invention first provides a rice blast-resistant receptor-like kinase OsLTK1 gene, the amino acid sequence encoded by the gene is shown in SEQ ID NO: 3.

[0007] The present invention provides a rice blast-resistant receptor-like kinase OsLTK1 gene, the nucleotide sequence of which is shown as SEQ ID NO: 1, or the CDS nucleotide sequence of which is shown as SEQ ID NO: 2.

[0008] The present invention further provides a protein encoded by the rice blast-resistant receptor-like kinase OsLTK1 gene, the amino acid sequence of which is shown in SEQ ID NO:3.

[0009] The present invention provides a method for improving the rice blast resistance, which comprises overexpressing the rice blast resistance receptor-like kinase OsLTK1 gene in rice, wherein the rice blast resistance receptor-like kinase OsLTK1 gene encodes a protein with the amino acid sequence shown in SEQ ID NO: 3.

[0010] Specifically, the overexpression method is: cloning the rice blast-resistant receptor-like kinase OsLTK1 gene and constructing a plant expression vector, transferring the plant expression vector into Agrobacterium, then inoculating rice, and selecting the overexpression strain or its offspring with rice blast resistance.

[0011] Preferably, the CDS nucleotide sequence of the rice blast-resistant receptor-like kinase OsLTK1 gene is shown as SEQ ID NO: 2.

[0012] More specifically, the plant expression vector is pXQ, and the Agrobacterium is EHA105.

[0013] The present invention provides the use of the rice blast-resistant receptor-like kinase OsLTK1 gene or the protein encoded by the rice blast-resistant receptor-like kinase OsLTK1 gene in cultivating rice varieties with enhanced blast-resistant ability.

[0014] The present invention also provides the use of the rice blast-resistant receptor-like kinase OsLTK1 gene in identifying rice varieties or plants with enhanced resistance to rice blast, wherein the rice varieties or plants with enhanced resistance to rice blast are obtained by the above method.

[0015] The present invention facilitates the cultivation of superior rice varieties, particularly disease-resistant varieties, and provides a basis for the subsequent screening of highly resistant rice varieties. For example, the present invention can provide rice overexpressing the receptor-like kinase OsLTK1, enabling the generation of even more resistant transgenic rice materials through hybridization. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The expression levels of the rice receptor kinase OsLTK1 gene after infection with the rice blast fungus and in panicles and leaves under normal conditions are analyzed. A represents the expression level of the OsLTK1 gene in leaves after pathogen stress; B represents the comparison of the expression levels of the OsLTK1 gene in panicles and leaves under natural growth conditions.

[0017] Figure 2 This figure shows the disease resistance of rice plants overexpressing and knocking out the rice receptor kinase OsLTK1. Figure A shows western blot analysis of OsLTK1 protein overexpression; Figure B shows the construction of the target sequence of the OsLTK1 knockout mutant using Crispr / Cas9 technology and the sequence alignment of the target gene knockout; Figure C shows the pathogenicity of the overexpressed rice plants after wounding; and Figure D shows the statistical comparison of lesion length after wounding inoculation. DETAILED DESCRIPTION

[0018] The present invention is described below through specific embodiments in order to better understand the present invention, but it does not constitute a limitation of the present invention.

[0019] Example 1: Analysis of rice receptor-like kinase OsLTK1 gene expression levels

[0020] The present invention uses 99-20 spore solution to inoculate rice leaves, and the spore solution concentration is 1×10 5 / mL, and then extracted the total RNA of the leaves at 0h, 12h, 24h, 36h, 48h, and 72h of infection for fluorescence quantitative analysis to detect the expression level of OsLTK1 gene. The results showed that the expression level of OsLTK1 gene (ABB47827.1) increased significantly at 36h ( Figure 1 Total RNA was extracted from rice panicles at the filling stage under natural growth conditions. The expression level of OsLTK1 gene was detected by fluorescence quantitative method and compared with the expression level of the gene in leaves. It was found that the expression level of OsLTK1 gene in panicles was significantly higher than that in leaves ( Figure 1 Middle B).

[0021] Example 2: Obtaining Rice Overexpressing the Rice Receptor Kinase OsLTK1 Gene

[0022] The present invention utilizes homologous recombination to construct the CDS nucleotide sequence (SEQ ID NO: 2) of the target gene OsLTK1 into the pXQ vector. When designing primers, homologous arms near the SmaI restriction site in the pXQ vector are first added to the upstream and downstream primers of OsLTK1 to amplify the OsLTK1 gene. The target gene and the linearized pXQ vector digested with SmaI are then ligated using a homologous recombination enzyme to construct the pXQ-OsLTK1 vector. Because the vector carries a Flag tag, it can be used for subsequent verification. The pXQ-OsLTK1 vector is then chemically transformed into Agrobacterium tumefaciens EHA105, and positive monoclonal colonies are selected to prepare OD values. 600 = 0.2 infection solution, infect rice callus with the infection solution for 15 minutes, and then culture the callus at 20℃ for 48-72 hours.

[0023] Subsequently, single-clone callus tissue was picked onto screening medium and cultured in the dark at 26°C. After 25 days, positive single-clone callus tissue was picked for subculture at 26°C in the dark. After 7-10 days of culture, the positive callus tissue was inoculated onto differentiation medium. After 15 days of culture under light at 27°C, the differentiated 3-5 cm buds were inoculated onto rooting medium and cultured at 30°C for 7 days.

[0024] When the seedlings were about 8 cm long, plants with well-developed root systems and normal growth were selected and cultured indoors for 7 days before being transferred to the field for seed propagation. After two seed propagations, plants overexpressing the OsLTK1 gene were obtained. From these transgenic plants, two overexpressing strains were selected and Western blotted to confirm the gene's overexpression in rice. Figure 2 (A) shows that the transgenic plants obtained can be used for subsequent experiments.

[0025] Example 3: Obtaining Rice with OsLTK1 Gene Knockout

[0026] The present invention utilizes Crispr / Cas9 technology to first construct an OsLTK1 knockout vector, and then transfers the knockout vector into Agrobacterium EHA105 by chemical transformation. The positive monoclonal colonies are picked to prepare the OD 600 = 0.2 infection liquid, the rice callus is infected with the infection liquid for 15 minutes, and then the callus is cultured at 20 ° C for 48-72 hours, then the single clone callus is picked out and placed on the screening medium, and screening and culture are carried out at 26 ° C in the dark. After 25 days, the positive single clone callus is picked out and subcultured at 26 ° C in the dark. After 7-10 days of culture, the positive callus is inoculated on the differentiation medium, and after 15 days of light culture at 27 ° C, the differentiated 3-5 cm buds are inoculated into the rooting medium and cultured at 30 ° C in the light for 7 days.

[0027] When the seedlings were about 8 cm long, plants with well-developed root systems and normal growth were selected and cultured indoors for 7 days before being transferred to the field for seed propagation. After two consecutive seed propagation and screening, a knockout mutant of the OsLTK1 gene was obtained. Target site sequencing confirmed that the gene had been effectively knocked out. The mutant exhibited a typical biallelic InDel mutation, specifically a deletion and a base insertion in the target sequence ( Figure 2 The availability of two mutant rice materials reduces the risk that the phenotypes are caused by off-target or random events, provides a reproducible chain of evidence for the experiment, and significantly improves the reliability of the conclusions.

[0028] Example 4: Analysis of the resistance and susceptibility of rice overexpressing and knockout rice expressing the rice receptor kinase OsLTK1 gene

[0029] The present invention multiplies the obtained overexpression and knockout transgenic rice plants to obtain second-generation stable genetic offspring plants for pathogenicity determination. The spores of the wild-type strain 99-20 of the rice blast fungus were inoculated by wounding the rice leaves. The concentration of the spores of the 99-20 strain was 8×10 5 / mL; The results showed that compared with the wild-type rice material ZH11, the OsLTK1 gene overexpression material showed disease resistance, while the knockout material showed disease susceptibility ( Figure 2 Further statistical analysis revealed that the lesion extension length of wild-type rice was 1.6 times that of the overexpression rice line, while the lesion extension length of the knockout rice line was 1.3 times that of the wild-type rice ( Figure 2 The above results indicate that the OsLTK1 gene plays an important role in the immune mechanism of rice against rice blast.

Claims

1. A method for improving the resistance of rice to rice blast fungus, characterized in that: Overexpression of the rice receptor kinase OsLTK1 gene in rice to obtain rice with improved resistance to rice blast fungus; The amino acid sequence encoded by the rice receptor kinase OsLTK1 gene is shown in SEQ ID NO:

3.

2. The method according to claim 1, characterized in that The genomic nucleotide sequence of the rice receptor kinase-like OsLTK1 gene is shown in SEQ ID NO: 1, or the CDS nucleotide sequence of the rice receptor kinase-like OsLTK1 gene is shown in SEQ ID NO:

2.

3. The method according to claim 1 or 2, characterized in that The overexpression method comprises: constructing a plant expression vector with the rice receptor kinase OsLTK1 gene, transferring the plant expression vector into Agrobacterium, inoculating rice, and selecting an overexpression strain or its offspring with improved rice blast resistance.

4. The method according to claim 3, characterized in that The plant expression vector is pXQ.

5. The method according to claim 3, characterized in that The Agrobacterium is EHA105.

6. Use of a rice receptor kinase-like OsLTK1 gene, or a protein encoded by it, in breeding rice varieties with enhanced resistance to rice blast fungus; The amino acid sequence encoded by the rice receptor kinase OsLTK1 gene is shown in SEQ ID NO:

3.

7. The use according to claim 6, characterized in that The genomic nucleotide sequence of the rice receptor kinase-like OsLTK1 gene is shown in SEQ ID NO: 1, or the CDS nucleotide sequence of the rice receptor kinase-like OsLTK1 gene is shown in SEQ ID NO:

2.

8. Use of a rice receptor kinase OsLTK1 gene in identifying rice varieties or plants with enhanced resistance to rice blast fungus, characterized in that: The amino acid sequence encoded by the rice receptor kinase OsLTK1 gene is shown in SEQ ID NO:

3.

9. The use according to claim 8, characterized in that The genomic nucleotide sequence of the rice receptor kinase-like OsLTK1 gene is shown in SEQ ID NO: 1, or the CDS nucleotide sequence of the rice receptor kinase-like OsLTK1 gene is shown in SEQ ID NO:

2.

10. The use according to claim 9, characterized in that The rice variety or plant with enhanced resistance to rice blast fungus is obtained by the method according to any one of claims 1 to 5.

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