Application of rice receptor-like kinase rlk19 in resistance to magnaporthe oryzae

Abstract

The application discloses application of rice receptor-like kinase RLK19 in regulating rice resistance to Magnaporthe oryzae. The application finds for the first time that overexpression of rice receptor-like kinase RLK19 positively regulates the resistance of rice to Magnaporthe oryzae, RLK19 affects sustained activation of a phosphorylation pathway MPK3 / MPK6 through phosphorylation, and induces expression of a disease resistance related gene, so as to regulate the resistance of rice to Magnaporthe oryzae. The application can be used for cultivating a plant variety resistant to Magnaporthe oryzae, and has application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of rice receptor kinase RLK19 in rice blast fungus resistance. Background Technology

[0002] Rice (Oryza sativa) is one of the most important food crops in the world. The population that eats rice as their staple food accounts for about 50% of the world's total population. Since 2011, under the combined effect of factors such as scientific and technological progress, the total area of ​​rice planting in my country has remained stable at about 30 million hectares per year, with a total output of more than 200 million tons. Therefore, ensuring the safe production of rice is of great significance to social and economic development. At present, rice blast, rice false smut and rice sheath blight are the three major diseases that harm rice in my country [2]. Among them, rice blast, caused by the filamentous pathogenic fungus Magnaphortheoryzae, is the most devastating rice disease and ranks first among fungal diseases. The rice blast fungus can be spread by air and water flow and can cause damage throughout the entire growth period of rice. Therefore, once it occurs, it will spread rapidly and have a serious impact on the yield of rice fields. In recent years, the spread of rice blast has invaded many rice-growing areas in the world, including Kenya, Italy and Bangladesh. As a major rice-producing country, my country has seen rice blast disease damage on an average annual scale of approximately 7.5 × 10⁹ hectares in recent years. It occurs in early, mid, and late-season rice varieties across all rice-growing regions of the country. Mild cases result in yield losses of 15-20%, more severe cases 40-50%, and extremely severe cases 70-90%, sometimes leading to complete crop failure. The annual loss of rice yield due to this disease amounts to billions of kilograms, and the disease's spread is showing a continuous upward trend. Currently, the control of rice blast mainly relies on the breeding of resistant varieties and the use of chemical pesticides. However, on the one hand, in actual field production, rice blast strains are prone to virulence mutations, producing new physiological races, causing newly bred resistant rice varieties to lose their resistance after only a few years of use. On the other hand, the overuse and irrational application of chemical pesticides not only pollute the environment but also threaten human and animal health and food safety. Therefore, elucidating the pathogenic mechanism of rice blast fungi and conducting in-depth research on the molecular mechanism of the interaction between rice blast fungi and the host are of great importance for the development of novel, low-toxicity, and highly effective fungicides against rice blast fungi.

[0003] Plants live in complex environments and are constantly threatened by various pathogens. To defend against these attacks, plants have evolved sophisticated strategies that translate into effective immune responses. The perception of conserved pathogen-associated molecular patterns (PAMPs) is typically mediated by pattern recognition receptors (PRRs) located on the cell surface. PAMP recognition leads to the rapid activation of a series of early immune responses, including the activation of the mitogen-activated protein kinase (MAPK) cascade and the production of reactive oxygen species (ROS) to combat pathogen infection. To overcome PAMP-triggered immunity (PTI), pathogens deliver specific factors called effector factors to the host to suppress PTI. In turn, plants have developed various resistance proteins to sense effectors and activate effector-triggered immunity, a response involving hypersensitivity (HR) to cell death.

[0004] Receptor-like kinases (RLKs) play a crucial role in sensing external stimuli, activating downstream signaling pathways, and regulating cellular responses to pathogen infection. Cysteine-rich receptor-like kinases (CRKs) are a subfamily of plant RLKs, exhibiting typical RLK structural features: an extracellular domain responsible for signal sensing, a single-pass transmembrane domain, and an intracellular serine / threonine protein kinase domain responsible for signal transduction. Most CRKs contain two copies of the Domain of Unknown Function 26 (DUF26) domain in their extracellular region. The DUF26 domain contains three conserved cysteine ​​residues with a C-X8-C-X2-C conformation, which may form potential targets for dithiol redox regulation. CRK genes play a vital role in regulating plant disease resistance and programmed cell death (PCD). In Arabidopsis, AtCRK5 and AtCRK13 are rapidly induced to express by Pseudomonas syringae. Overexpression of AtCRK5 or AtCRK13 induces cellular PCD, and the rapid expression of pathogenesis-related proteins (PR) genes in plants enhances resistance to Pseudomonas syringae [6-7]. Overexpression of AtCRK4, AtCRK19, and AtCRK20, which are homologous to AtCRK5, also leads to PCD. Overexpression of AtCRK4, AtCRK6, and AtCRK36 triggers an immune response triggered by the pathogen-associated molecular pattern (PAMP), which also enhances plant resistance to Pseudomonas syringae. AtCRK7 is involved in mediating the production of extracellular reactive oxygen species. The wheat TaCRK1 gene regulates wheat resistance to *Rhizoctonia graminearum*; in rice, CRK10 participates in OsNPR1-mediated systemic acquired resistance; in sea island cotton, GbCRK18 participates in resistance to *Verticillium dahliae* and affects cotton resistance to *Verticillium wilt*; and the wheat CRK gene Stb16q exhibits broad-spectrum resistance to *Trichoderma viride*. These studies indicate that CRK genes play an important role in the regulation of plant disease resistance. Currently, 45 CRK genes have been reported in rice; however, their roles in rice disease defense remain unclear. Summary of the Invention

[0005] This invention provides a theoretical basis for the role of CRK genes in rice resistance to rice blast fungus by studying the potential CRK gene RLK19 in rice. This is expected to provide high-quality gene resources for rice disease resistance breeding. Simultaneously, molecular cloning techniques are used to screen and cultivate disease-resistant, high-yielding rice plants. Therefore, the main objective of this invention is to provide a method for improving the resistance of rice to rice blast fungus.

[0006] The present invention first provides a rice receptor kinase RLK1 gene, the amino acid sequence of which is shown in SEQ ID NO:2.

[0007] This invention provides a rice receptor kinase RLK1 gene, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0008] The present invention further provides a protein encoded by the rice receptor kinase RLK1 gene, the amino acid sequence of which is shown in SEQ ID NO:2.

[0009] This invention provides a method for improving plant disease resistance, which involves overexpressing the rice receptor kinase RLK1 gene in plants. The rice receptor kinase RLK1 gene is a gene encoding a protein with the amino acid sequence shown in SEQ ID NO:3. The plant is a monocotyledonous plant, preferably rice or tobacco. The disease resistance refers to resistance to plant diseases caused by rice blast fungus or Phytophthora blight.

[0010] In a specific implementation, the overexpression method is as follows: transfecting or introducing the rice receptor kinase RLK1 gene into plants, and selecting overexpression lines or their offspring with enhanced disease resistance.

[0011] Furthermore, the rice receptor kinase RLK1 gene is constructed into the plant expression vector pXQ and then introduced into plants, for example, through Agrobacterium infection.

[0012] This invention provides the application of the rice receptor kinase RLK1 gene, or the protein encoded by the rice receptor kinase RLK1 gene, in the cultivation of plant varieties with enhanced disease resistance.

[0013] Preferably, the plant is a monocotyledonous plant, and more preferably, the plant is rice or tobacco; the disease resistance refers to resistance to plant diseases caused by rice blast fungus or Phytophthora blight.

[0014] This invention relates to the application of the potential receptor-like kinase RLK19 in rice disease resistance. Transgenic plants overexpressing RLK19 exhibit significant resistance to rice blast fungus during infection. Furthermore, during interaction with the rice blast fungus, the phosphorylation pathway MPK3 / MPK is continuously activated, thereby significantly enhancing resistance to the fungus. The transgenic plants involved in this invention do not affect normal growth and fruit setting. Attached Figure Description

[0015] Figure 1 The RLK19 gene response to rice blast fungus infection is represented by the changes in the transcription level of the RLK19 gene at different time points after rice ZH11 was inoculated with rice blast fungus.

[0016] Figure 2 To verify the expression level of RLK19 gene overexpression in transgenic plants.

[0017] Figure 3 To determine the pathogenicity of RLK19-based transgenic plants against rice blast fungus.

[0018] Figure 4 To verify that RLK19 is a potential receptor-like kinase of the CRK family. In this context, A indicates that RLK19 possesses protein domains characteristic of the typical CRK family; B indicates that RLK19 is located on the cell membrane in tobacco; and C indicates that RLK19 is located on the cell membrane in rice protoplasts.

[0019] Figure 5 This study aimed to determine the sustained activation of MPK3 / MPK6 in the RLK19 phosphorylation pathway.

[0020] Example 1: The RLK19 gene plays an important role in the early stage of rice resistance to rice blast infection.

[0021] In our preliminary laboratory experiments, we obtained a batch of potential RLK / RLCK genes using mass spectrometry. We focused on some genes possessing the DUF26 domain. After quantitative analysis, we found that RLK19 plays a role in the early stages of rice blast fungus infection. Therefore, this invention focuses on the RLK19 gene. In this invention, rice ZH11 was infected with the wild-type strain Guy11 of rice blast fungus. Total RNA was extracted from rice at 0h, 12h, 24h, 36h, and 48h. The extracted RNA was reverse transcribed to synthesize cDNA, which was used to detect the expression level of the RLK19 gene (its nucleotide sequence is shown in SEQ ID No: 1, and the encoded amino acid sequence is shown in SEQ ID No: 2). RNA was extracted from rice leaves at different infection stages using an RNA extraction kit from Tiangen Biotech. Reverse transcription was performed using a TaKaRa reverse transcription kit. Quantitative real-time PCR was then performed after reverse transcription. This invention discovered that after infection by *Strombus oryzae*, the transcriptional level of RLK19 was significantly upregulated at both 24 and 36 hours after infection, especially at 24 hours, confirming that the RLK19 gene plays an important role in the early stage of rice resistance to *Strombus oryzae* infection. Figure 1 ).

[0022] SEQ ID No: 1:

[0023]

[0024] SEQ ID No: 2:

[0025] MAILTVLPLVLVMLLLPLAAIASGDPPWQHYCGSSGNYTAGSKYQANLQALAATLPSTASSSSPALFAKDAAGGGDAEPDRVFALTLCRGDTASANASSSSCADCASRAFRDAQSVCPYSKEVAVYYDPCLLYFSGDDFLSSPANPAQVRLYDVDRSTRRGGGGADFVTLVRALLSYTMQWAVPYNSTGGGAAAVRWYTTVRMDVVTPPLFSLMQCTPDMSGGDCRQCLQDLVGNTTFNGSVSGVRNIGARCGYRYDTYKFYGGEPKLKIGSLSEINSTAPSSPPPPPPVTETRSGRKKVLTVALLVPLIALCPVVIFCFAWIRRLRNHKSMLRKKDTMAREEVLKLWRLEESDSEFMLFDFSQIEDATSNFSEDKKLGEGGFGSVYKGQLPNGLEVAVKRLAAHSSQGLVEFKNEIQLIAKLQHTNLVNLRGCCIQGEENLLIYEYMPNKSLDFFIFDLKRAALLNWKTRLNIIEGITQGLLYLHKHSRLCIIHRDLKASNILLDRDMNPKISDFGLAKIFDSNDVQRNTKRVVGTYGYMAPEYASEGCFSLKSDVFSFGVLVLEIISGKRNAGFHQYGDFFNLLGYAWQLWKDGSWHELVDPSLVSEGQMMEIKKCMKVALLCVQENAVDRPTMSAVVKMLSSELKILPEPKQPAFFNVRVKHGELSNTAPSSINDVTITIVNGR。

[0026] Example 2: Obtaining and Pathogenicity Determination of RLK19 Gene Overexpression Plants

[0027] This invention also utilizes an enzyme digestion and ligation method to construct the target gene RLK19 into the PXQ-flag vector. During primer design, SmaI restriction sites were added to the upstream and downstream primers for RLK19 to amplify the RLK19 gene. The target gene and vector were then linearized by restriction endonuclease SmaI. The vector and fragment were ligated using homologous recombination ligase to construct the PXQ-RLK19-flag vector. The vector was constructed in our laboratory and transformed by Aidijing Biotechnology Co., Ltd. to obtain plants overexpressing the RLK19 gene. The expression level of the RLK19 gene in the obtained transgenic plants was first quantitatively analyzed by qRT-PCR, demonstrating that the overexpression of RLK19 was not problematic. Figure 2 ).

[0028] This invention propagates the obtained overexpressing rice plants to obtain third-generation stably inherited progeny plants for pathogenicity testing. Using the punch inoculation technique, spores of the wild-type rice blast fungus Guy11 were inoculated onto rice leaves at a concentration of 5 x 10⁴ spores / ml. It was found that the lesions in rice overexpressing RLK19 were significantly smaller than those in wild-type rice ZH11. Figure 3 This indicates that RLK19 is regulating rice resistance to rice blast and improving rice's disease resistance.

[0029] Example 3: RLK19 is a potential receptor-like kinase family CRK.

[0030] This invention, through protein structure prediction, revealed that RLK19 contains two unknown functional domains (DUF26 domain, each with two copies in the extracellular region), a single-pass transmembrane domain, and an intracellular serine / threonine protein kinase domain responsible for signal transduction. Figure 4 (A). This invention utilizes a homologous recombinase to construct a fusion expression vector PBIN-RLK19-GFP, injects it into tobacco for expression, and further treats tobacco cells with a high concentration of sucrose solution to induce plasmolysis. It was found that RLK19-GFP localizes along the cell membrane infolding, indicating its localization on the cell membrane, consistent with potential RLK characteristics. Figure 4 (Middle B). This invention further constructed a PXQ-RLK19-GFP fusion expression vector and transformed it into rice protoplasts. Simultaneously, using the cell membrane dye FM4-64 to stain the rice protoplasts, it was found that the fluorescence produced by RLK19-GFP co-localized with the cell membrane fluorescence stained by FM4-64. Figure 4 The presence of C indicates that RLK1 is located on the rice cell membrane, further demonstrating that RLK19 may be a potential CRK family located on the cell membrane.

[0031] Example 4: RLK19 persistently activates the phosphorylation pathway MPK3 / MPK6 pathway

[0032] This invention utilizes rice overexpressing ZH11 and RLK19 to infect rice with *Bacillus oryzae*, and extracts total protein from the rice at 0h, 12h, 24h, 36h, and 48h for Western blotting experiments. This invention uses the MPK3 / MPK6 antibody P-p44 / 42 to detect protein kinase expression, and performs Ponceau S staining on the protein samples to ensure consistent protein loading. Compared to ZH11, RLK19-overexpressing rice showed sustained activation of MPK3 / MPK6 phosphorylation at 36 and 48h, indicating that RLK19 can significantly enhance resistance to *Bacillus oryzae* by continuously activating phosphorylation. Figure 5 ).

Claims

1. A method for improving the resistance of rice to rice blast fungus, characterized in that, The rice receptor kinase RLK19 gene was overexpressed in rice. The rice receptor kinase RLK19 gene is a gene encoding a protein with the amino acid sequence shown in SEQ ID NO:

2.

2. The method according to claim 1, characterized in that, The overexpression method is as follows: the rice receptor kinase RLK19 gene is transfected or introduced into rice, and overexpression lines or their progeny that show increased resistance to rice blast fungus are selected.

3. The method according to claim 1 or 2, characterized in that, The cDNA nucleotide sequence of the rice receptor kinase RLK19 gene is shown in SEQ ID NO:

1.

4. The method according to claim 3, characterized in that, The rice receptor kinase RLK19 gene was constructed into the plant expression vector pXQ and then introduced into rice.

5. The method according to claim 4, characterized in that, Introduced into rice through infection with Agrobacterium.

6. The application of the rice receptor kinase RLK19 gene, or the protein encoded by the rice receptor kinase RLK19 gene, in the breeding of rice varieties with enhanced resistance to rice blast fungus. The rice receptor kinase RLK19 gene is a gene encoding a protein with the amino acid sequence shown in SEQ ID NO:2.

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