Rice receptor kinase OsLTK1 and its application in rice blast fungus resistance

Abstract

This invention belongs to the field of biotechnology and discloses a rice receptor-like kinase OsLTK1 and its application in resistance to rice blast fungus. This invention is the first to discover that the receptor-like kinase OsLTK1 positively regulates rice resistance to rice blast fungus. Further research shows that transgenic plants overexpressing the OsLTK1 gene in rice exhibit significant resistance to rice blast fungus. Therefore, the OsLTK1 gene can be used for the breeding of rice varieties resistant to rice blast fungus and has significant application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a rice receptor kinase OsLTK1 and its application in rice blast fungus resistance. Background Technology

[0002] Rice, as a major grain crop in my country, accounts for approximately one-quarter of the country's total grain planting area. However, rice diseases caused by Magnaphalthe oryzae severely impact normal rice production, reducing rice quality and commercial value. Rice blast, along with rice sheath blight, rice false smut, and rice viral diseases, is considered one of the four major rice diseases. Rice blast occurs in both northern and southern rice-growing areas of my country, causing reduced rice yields. Currently, the control of rice blast mainly relies on developing new resistant rice varieties, supplemented by cultivation practices and chemical control. However, the rice blast fungus is highly prone to mutation, causing resistant rice varieties to often lose their resistance within a few years of introduction. Furthermore, chemical control methods are too simplistic and often fail to address the root cause of the problem; their extensive use also leads to environmental pollution. Therefore, a deeper understanding of the molecular mechanisms of the interaction between rice and rice blast fungus is not only helpful in developing and applying broad-spectrum and effective control strategies against rice blast fungus, but also provides a theoretical basis for exploring new gene resources for breeding rice blast fungus, which has important application value for the sustainable production of rice in the future.

[0003] Plants have evolved two immune mechanisms to defend against pathogen infection: the first is pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), and the second is effector-triggered immunity (ETI). When plants are attacked by external pathogens, specific receptors (PRRs) on the plant surface... S It can recognize PAMPs on the surface of pathogens. SAt this point, the plant's PTI immune mechanism is activated, inducing a series of immune responses to inhibit pathogen infection. Through long-term 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 pathogen infection, disease resistance-related proteins in plant cells can specifically recognize certain effector factors and trigger the plant's second immune mechanism (ETI) [Bigeard I J, Colcombet J, Hirt H. Signaling mechanisms in pattern-triggered immunity (PTI) [J]. Mol Plant, 2015, 8(4): 521-39.;Jones JD, Dangl J 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 relevant disease resistance factors, generate reactive oxygen species (ROS), and induce callose deposition during the process of limiting the invasion of external pathogens. ROS can directly kill invading pathogens and also act as signaling molecules to activate downstream immune responses, thus enhancing plant resistance. Studies have found that plant ETI immune mechanisms can enhance the expression of NADPH oxidase RBOHD, while cytoplasmic receptor kinases in PTI immune mechanisms 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.]. Furthermore, PTI triggered by PAMPs is stable, persistent, and broad-spectrum; explaining its mechanism has significant theoretical and practical value for improving the long-lasting disease resistance of crops. Receptor-like kinases (RLKs) are a type of pattern recognition receptors (PRRs) on the plant surface. They are one of the largest transmembrane signaling protein families in plants. After sensing external signals in their extracellular domains, they transmit signals through their intracellular kinase domains, regulating plant growth and development, immune defense, and stress responses.The typical structure of RLKs includes an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular kinase. Based on the characteristics of the extracellular domain, RLKs are divided into more than 20 subfamilies, such as LRR-RLK and Lys-RLK. More than 600 members have been identified in Arabidopsis thaliana [Shiu SH, Bleecker A B. Plant receptor-like kinase genefamily: diversity, function, and signaling [J]. Science's STKE : signaltransduction knowledge environment, 2001, 2001(113): re22.]. In recent years, some studies have found that RLK proteins play a very important role in the resistance of rice to rice blast infection. Currently, 17 RLK proteins have been identified in rice that are involved in resistance to rice blast fungus. For example, OsBDR1 negatively regulates rice resistance to rice blast fungus by interacting with mitogen-activated protein kinase 3 (MPK3) to inhibit the jasmonate signaling pathway and terpenoid biosynthesis [Wang L, Xu G, Li L, et al. The OsBDR1-MPK3 module negatively regulates blast resistance by suppressing the jasmonate signaling and terpenoid biosynthesis pathway [J]. Proc Natl Acad Sci USA, 2023, 120(13): e2211102120.]. Furthermore, salicylic acid (SA) and jasmonic acid (JA) accumulate significantly in ALS-overexpressing rice, resulting in a markedly enhanced resistance to rice blast fungus. In terms of ROS regulation, the SDS2 protein actively regulates immune responses by phosphorylating the NADPH oxidase OsRbohB through interaction with OsRLCK118 and OsRLCK176, thereby stimulating ROS production [Fan J, Bai P, Ning Y, et al. The Monocot-Specific Receptor-like Kinase SDS2 Controls Cell Death and Immunity in Rice [J]. 2018: 498-510.e5.]. OsRLCK118 enhances rice resistance to rice blast fungus by affecting ROS levels and defense hormone-related signaling pathways; silencing OsRLCK118 increases susceptibility to rice blast 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 crucial for maintaining chlorophyll content in rice leaves. Loss of PWL1 function leads to reduced chlorophyll levels and premature senescence [Xu J, Wang C, Wang F, et al. PWL1, a G-type lectin receptor-like kinase, positively regulates leaf senescence and heat tolerance but negatively regulates resistance to Xanthomonas oryzae in rice [J]. Plantbiotechnology journal, 2023, 21(12): 2525-45.]. 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 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 tiller-related genes [Zhao J, Wu C, Yuan S, et al. Kinase activity of OsBRI1 is 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 rice varieties that are high-yielding and disease-resistant, and identifying genes that can precisely regulate rice growth and disease resistance at specific growth stages or in specific tissues holds promise for solving this problem. The rapid development of genome sequencing and bioinformatics analysis technologies has enabled the identification of more RLK genes in rice, making it possible to further study the biological functions and molecular mechanisms of RLK proteins, which is beneficial for breeding new disease-resistant and high-yielding rice varieties through molecular breeding. Summary of the Invention

[0005] This invention reveals that transgenic rice overexpressing the OsLTK1 gene exhibits significant resistance to rice blast fungus. OsLTK1 gene knockout mutations result in susceptible rice phenotypes, while the growth of knockout rice materials is not affected. This leads to the completion of this invention.

[0006] The present invention first provides a receptor kinase OsLTK1 gene for rice resistance to rice blast fungus, the amino acid sequence of which is shown in SEQ ID NO:3.

[0007] This invention provides a receptor kinase OsLTK1 gene for rice resistance to rice blast fungus, the nucleotide sequence of which is shown in SEQ ID NO:1, or the CDS nucleotide sequence of which is shown in SEQ ID NO:2.

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

[0009] This invention provides a method for improving the resistance of rice to rice blast fungus, which involves overexpressing the rice blast fungus receptor kinase OsLTK1 gene in rice. The rice blast fungus receptor kinase OsLTK1 gene is a gene encoding a protein with the amino acid sequence shown in SEQ ID NO:3.

[0010] Specifically, the overexpression method is as follows: the OsLTK1 gene, a receptor kinase similar to rice blast fungus, is cloned and a plant expression vector is constructed. The plant expression vector is then transferred into Agrobacterium, and rice is inoculated. Overexpression lines or their progeny with rice blast resistance are selected.

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

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

[0013] This invention provides the application of the OsLTK1 gene, a receptor kinase-like gene for rice resistance to rice blast fungus, or the protein encoded by the OsLTK1 gene for rice resistance to rice blast fungus, in the cultivation of rice varieties with enhanced resistance to rice blast fungus.

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

[0015] This invention is beneficial for the breeding of superior rice varieties, especially disease-resistant varieties, and provides a basis for the later screening of highly resistant rice varieties. For example, this invention can provide rice that overexpresses the receptor kinase OsLTK1, and through hybridization, obtain transgenic rice materials with higher resistance. Attached Figure Description

[0016] Figure 1 This study analyzed the expression levels of the rice receptor kinase OsLTK1 gene in rice after infection with rice blast fungus, as well as in the panicle and leaves under normal conditions. In this study, A represents the expression level of the OsLTK1 gene in leaves after pathogen stress; B represents a comparison of the expression levels of the OsLTK1 gene in the panicle and leaves under natural growth conditions.

[0017] Figure 2 This study aims to detect the disease resistance of rice plants overexpressing and knocking out the OsLTK1 receptor kinase gene. In the table, A represents Western blotting of OsLTK1 protein overexpressing plants; B represents the construction of the target sequence for the OsLTK1 gene knockout mutant obtained using CRISPR / Cas9 technology and the sequence alignment results of the knockout gene; C represents the pathogenicity results of wound-induced inoculation in overexpressing rice; and D represents the statistical analysis and comparison of lesion length after wound-induced inoculation. Detailed Implementation

[0018] The present invention will be described below through specific embodiments in order to better understand the present invention, but this does not constitute a limitation on the present invention.

[0019] Example 1: Analysis of the expression level of rice receptor kinase OsLTK1 gene

[0020] This invention uses 99-20 spore culture to inoculate rice leaves, with a spore concentration of 1×10⁻⁶. 5 The total RNA was extracted from leaves at 0h, 12h, 24h, 36h, 48h, and 72h after infection for quantitative fluorescence analysis to detect the expression level of the OsLTK1 gene. The results showed that the expression level of the OsLTK1 gene (ABB47827.1) increased significantly at 36h. Figure 1 (A); Total RNA was extracted from the panicle of rice during the grain-filling stage under natural growth conditions. The expression level of the OsLTK1 gene was detected by quantitative real-time analysis and compared with the expression level of the gene in the leaves. It was found that the expression level of the OsLTK1 gene in the panicle was significantly higher than that in the leaves. Figure 1 (B)

[0021] Example 2: Obtaining rice with OsLTK1 gene overexpression

[0022] This invention utilizes homologous recombination to construct the CDS nucleotide sequence (SEQ ID NO: 2) of the target gene OsLTK1 into a pXQ vector. During primer design, homologous arms near the SmaI restriction site in the pXQ vector were first added to the upstream and downstream primers of OsLTK1 to amplify the OsLTK1 gene. Then, the target gene and the linearized pXQ vector digested with SmaI were ligated using homologous recombination enzyme to form the pXQ-OsLTK1 vector. Because the vector contains a built-in flag tag, it can be used for later validation. The pXQ-OsLTK1 vector was transformed into Agrobacterium EHA105 via chemical transformation, and positive single colonies were selected to prepare OD. 600 Rice callus was infected with a bacterial solution of 0.2 for 15 min, and then cultured at 20°C for 48-72 h.

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

[0024] When the seedlings reached approximately 8cm in length, plants with well-developed root systems and normal growth were selected and cultured indoors for 7 days before being transferred to the field for propagation. After two propagation cycles, plants overexpressing the OsLTK1 gene were obtained. From these transgenic plants, two overexpression lines were selected, and Western spectroscopy was used to detect the overexpression of this gene in rice. Figure 2 (A) This proves that the obtained transgenic plants can be used for subsequent experiments.

[0025] Example 3: Obtaining rice with OsLTK1 gene knockout, a rice receptor kinase.

[0026] This invention utilizes CRISPR / Cas9 technology to first construct an OsLTK1 knockout vector, then transforms the knockout vector into Agrobacterium EHA105 via chemical transformation, and selects positive single colonies to prepare OD. 600 Rice callus was infected with a 0.2% inoculum solution for 15 minutes, and then cultured at 20°C for 48-72 hours. Subsequently, single-clone callus tissue was picked and transferred to a selection medium for selection culture at 26°C in the dark. After 25 days, positive single-clone callus tissue was picked and subcultured at 26°C in the dark. After 7-10 days of culture, the positive callus tissue was inoculated onto a differentiation medium and cultured at 27°C under light for 15 days. Then, the differentiated buds of 3-5 cm were inoculated onto a rooting medium and cultured at 30°C under light for 7 days.

[0027] When the seedlings reached approximately 8 cm in length, plants with well-developed root systems and normal growth were selected and cultured indoors for 7 days before being transferred to the field for propagation. After two rounds of propagation and selection, a knockout mutant of the OsLTK1 gene was obtained. Target sequencing confirmed that the gene had been effectively knocked out. The mutant exhibited a typical biallelic InDel mutation, specifically involving the deletion of the target sequence and the insertion of bases. Figure 2 (B) Obtaining two mutant rice materials reduces the risk that the phenotype is 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 resistance and susceptibility between rice varieties overexpressing and knocking out the rice receptor kinase OsLTK1 gene

[0029] This invention involves propagating the obtained overexpression and knockout transgenic rice plants to obtain second-generation stably inherited progeny plants for pathogenicity testing. The spores of the wild-type strain 99-20 of *Bacillus oryzae* were inoculated into rice leaves through leaf wounding, with a spore concentration of 8 × 10⁻⁶. 5 The results showed that, compared with the wild-type rice material ZH11, the OsLTK1 gene overexpression material was resistant to disease, while the knockout material was susceptible to disease. Figure 2 Further analysis revealed that the lesion extension length in wild-type rice was 1.6 times that in overexpressing rice lines, while the lesion extension length in knockout rice lines was 1.3 times that in wild-type rice. Figure 2 (D); The above results indicate that the OsLTK1 gene plays an important role in the immune mechanism of rice resistance to 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 was used to obtain rice 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.

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

2.

3. The method according to claim 1 or 2, characterized in that, The overexpression method is as follows: construct a plant expression vector from the rice receptor kinase OsLTK1 gene, transform the plant expression vector into Agrobacterium, then inoculate rice, and select overexpression lines or their progeny with improved resistance to rice blast.

4. The method according to claim 3, characterized in that, The plant expression vector was obtained by constructing the rice receptor kinase OsLTK1 gene into the pXQ vector.

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

6. Application of overexpression of rice receptor kinase OsLTK1 gene 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 application according to claim 6, characterized in that, The genomic nucleotide sequence of the rice receptor kinase OsLTK1 gene is shown in SEQ ID NO:1, or the CDS nucleotide sequence of the rice receptor kinase OsLTK1 gene is shown in SEQ ID NO:2.

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