Application of rice lipid transporter LTPL113 in fungal disease resistance

Abstract

The invention discloses an application of a rice lipid transporter protein LTPL113 in resisting fungal diseases. According to the invention, it is found for the first time that the OsLTPL113 gene participates in pollen outer wall development of rice and rice immune response by combining phosphatidic acid (PA) and phosphatidylserine (PS), including reactive oxygen species (ROS) bursting, so that the OsLTPL113 gene plays a positive regulation role in resistance of rice to Magnaporthe oryzae and Rhizoctonia solani. The invention further relates to knockout of the OsLTPL113 gene in rice and acquisition of an overexpression plant, and the broad-spectrum resistance of the overexpression plant to rice fungi is improved.

Description

Technical Field

[0001] This invention relates to the fields of plant molecular biology and crop genetic improvement, specifically to the application of a rice lipid transporter LTPL113 in resistance to fungal diseases, and the disease-resistant transgenic material constructed therefrom. Background Technology

[0002] Rice (Oryza sativa) is one of the world's major food crops, and various fungal diseases (such as rice blast and sheath blight) cause severe yield losses every year. Traditional control methods, such as chemical pesticides and the promotion of disease-resistant varieties, have problems such as rapid loss of resistance, environmental pollution, and limited varietal resources. Therefore, developing new disease-resistant genes and constructing stable, highly disease-resistant varieties through molecular breeding has become an urgent technical challenge.

[0003] Lipid transport proteins (LTPs) are a class of small, cysteine-rich proteins with hydrophobic channels that accommodate lipid ligands. LTPs perform a variety of functions in plants, participating not only in membrane homeostasis, cell wall synthesis, and signal transduction, but also playing important roles in biotic and abiotic stress responses, plant growth and development, seed storage, and germination (Missaoui K, Gonzalez-Klein Z, Pazos-Castro D, et al. Plant non-specific lipid transfer proteins: An overview [J]. Plant Physiology and Biochemistry, 2022, 171: 115-127). LcLTP1, as a positive immunomodulatory factor in litchi, is disrupted by the effector protein pectin acetylesterase PlPAE5 of *Pseudomonas litchiensis*, inhibiting its mediated salicylic acid immune signaling pathway (Situ J, Song Y, Feng D, et al. Oomycete pathogen pectin acetylesterase targets host lipid transfer protein to reduce salicylic acid signaling [J]. Plant Physiology, 2024, 194(3): 1779-1793.). Potato StLTP10 interacts with the ABA receptor PYL4, enhancing resistance to *Phytophthora infestans* by inducing stomatal closure (Wang C, Gao H, Chu Z, et al. Anon-specific lipid transfer protein, StLTP10, mediates resistance to *Phytophthora infestans* in potato [J]. Molecular Plant Pathology, 2021, 22(1):48-63).LTP protein OsAAI1 enhances plant drought resistance by strengthening reactive oxygen species scavenging capacity (Long Q, Qiu S, Man J, et al. OsAAI1 increases rice yield and drought tolerance dependent on ABA-mediated regulatory and ROS scavenging pathway [J]. Rice, 2023, 16(1:35.). OsLTPL159 is significantly upregulated under drought and salt stress, and overexpression of this gene significantly enhances plant environmental tolerance (Zhao J, Wang S, Qin J, et al. The lipid transfer protein OsLTPL159 is involved in cold tolerance at the early seedling stage in rice [J]. PlantBiotechnology Journal, 2020, 18(3): 756-769.).

[0004] Although the functions and mechanisms of action of some LTP proteins have been reported, the roles of most rice LTPs in fungal disease defense remain unclear. Therefore, identifying and cloning resistance-related LTPs is of significant guiding value for further understanding rice disease resistance mechanisms and exploring rice disease resistance gene resources. OsLTPL113 can provide a novel gene resource for constructing highly stable, broad-spectrum disease-resistant rice. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention has discovered that transgenic rice overexpressing the OsLTPL113 gene exhibits significant resistance to rice blast fungus and rice sheath blight pathogens. Conversely, rice with OsLTPL113 gene knockout mutations displays a susceptible phenotype. This invention is thus completed.

[0006] The technical solution adopted by this invention to solve its technical problem is:

[0007] In a first aspect, the present invention provides a rice lipid transporter protein OsLTPL113, said protein being selected from any one of the following (A1)-(A3):

[0008] (A1) A protein with the amino acid sequence shown in SEQ ID NO:3;

[0009] (A2) Proteins derived from 1) with the same activity, but with one or more amino acids substituted, deleted, and / or added to the amino acid sequence shown in SEQ ID NO.3;

[0010] (A3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of (A1) or (A2).

[0011] In a second aspect, the present invention provides a rice lipid transporter OsLTPL113 gene, which is selected from any one of the following (A1)-(A3):

[0012] (A1) The nucleotide sequence is shown in SEQ ID NO:1;

[0013] (A2) The CDS sequence is shown in SEQ ID NO:2;

[0014] (A3) A nucleotide sequence that hybridizes with a DNA sequence defined by (A2) under strict conditions.

[0015] Specifically, the amino acid sequence of the protein encoded by the rice lipid transporter OsLTPL113 gene is shown in SEQ ID NO:3.

[0016] Thirdly, the present invention provides a recombinant expression vector, expression cassette, transgenic cell line or genetically engineered bacteria containing the rice lipid transporter OsLTPL113 gene described above.

[0017] Fourthly, the present invention provides a method for improving the resistance of rice to fungal diseases, which involves overexpressing the rice lipid transporter OsLTPL113 gene in rice, wherein the rice lipid transporter OsLTPL113 gene is a gene encoding a protein with the amino acid sequence shown in SEQ ID NO:3.

[0018] Specifically, the overexpression method is as follows: the rice lipid transporter OsLTPL113 gene is cloned and a plant expression vector is constructed. The plant expression vector is then transformed into Agrobacterium, and rice is inoculated. Overexpression lines or their progeny with rice blast resistance are selected.

[0019] Preferably, the CDS sequence of the rice blast-resistant lipid transporter OsLTPL113 gene is shown in SEQ ID NO:2.

[0020] More specifically, the plant expression vector is pXQ, and the Agrobacterium is EHA105.

[0021] Fifthly, the present invention provides the application of the rice lipid transporter OsLTPL113 described above, the rice lipid transporter OsLTPL113 gene described above, or the recombinant expression vector, expression cassette, transgenic cell line or genetically engineered bacteria described above in rice resistance to fungal diseases or in the preparation of rice products resistant to fungal diseases.

[0022] Sixthly, the present invention provides the application of the rice lipid transporter OsLTPL113 described above, the rice lipid transporter OsLTPL113 gene described above, or the recombinant expression vector, expression cassette, transgenic cell line or genetically engineered bacteria described above in the cultivation of rice varieties with enhanced resistance to fungal diseases.

[0023] In a seventh aspect, the present invention also provides the rice lipid transporter OsLTPL113, the rice lipid transporter OsLTPL113 gene described above, or the recombinant expression vector, expression cassette, transgenic cell line or genetically engineered bacteria described above, in the identification of 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 methods.

[0024] Preferably, the fungus is Magnaphalthe oryzae, the rice blast fungus, and / or Rhizoctonia solani, the sheath blight fungus.

[0025] This invention is beneficial for the breeding of disease-resistant rice varieties and provides a basis for the later screening of highly resistant rice varieties. For example, this invention can provide rice varieties that overexpress the lipid transporter OsLTPL113, and through hybridization, obtain transgenic rice materials with higher resistance. Attached Figure Description

[0026] Figure 1 This study validates the overexpression and knockout of the rice lipid transporter OsLTPL113 gene in rice. 1A shows the construction of the target sequence for the OsLTPL113 gene knockout mutant obtained using CRISPR / Cas9 technology and the sequencing results of the knockout gene. 1B shows the Western blot validation of OsLTPL113 protein overexpression plants.

[0027] Figure 2 This study investigated the disease resistance of the lipid transporter OsLTPL113. 2A shows the resistance and susceptibility test results of OsLTPL113 gene mutant rice and overexpressing rice to *Strombus oryzae*, and 2B presents the statistical results of *Strombus oryzae* lesion length in 2A. 2C shows the resistance and susceptibility test results of OsLTPL113 gene mutant rice and overexpressing rice to *Sheath blight*, and 2D presents the statistical results of *Strombus oryzae* lesion length in 2C. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to the embodiments. Unless otherwise specified, all reagents or instruments used are considered to be conventional products that can be purchased on the market.

[0029] Example 1: Obtaining rice with overexpression of the rice receptor kinase OsLTPL113 gene

[0030] This invention utilizes homologous recombination to construct the target gene OsLTPL113 (CDS sequence shown in SEQ ID NO: 2) into the pCAMBIA2300 vector. First, a vector with the OsLTPL113 flag tag is obtained. Next, primers are designed, and homologous arms near the SmaI restriction site in the pCAMBIA2300 vector are added to the upstream and downstream primers of OsLTPL113-Flag, respectively, to amplify the OsLTPL113-Flag gene. Gene amplification uses Novizan high-fidelity enzyme (P510), and the experimental system follows the enzyme solution instructions. Then, the target gene and the linearized pCAMBIA2300 vector digested with SmaI are ligated using Novizan homologous recombinase (C115), with the ligation system following the enzyme solution instructions, completing the construction of the pCAMBIA2300-OsLTPL113-Flag vector. The flag tag of the vector can be used for later validation.

[0031] The pCAMBIA2300-OsLTPL113 vector was transformed into Agrobacterium EHA105 via chemical transformation, and positive single colonies were selected to prepare OD. 600 Rice ZH11 callus was infected with a 0.2% inoculum solution for 15 min, followed by incubation at 20℃ for 48-72 h. Single-clone callus tissue was then selected and cultured on a selection medium at 26℃ in the dark. After 25 days, positive single-clone callus tissue was selected and subcultured at 26℃ in the dark. After 7-10 days of culture, the positive callus tissue was inoculated onto a differentiation medium and cultured at 27℃ under light for 15 days. Then, 3-5 cm differentiated shoots were inoculated onto a rooting medium and cultured at 30℃ under light for 7 days. 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 propagation processes, plants overexpressing the OsLTPL113 gene were obtained.

[0032] From the obtained transgenic plants, we selected three overexpression lines and used Western spectroscopy to detect the overexpression of this gene in rice. Figure 1 (B) This proves that the obtained transgenic plants can be used for subsequent experiments.

[0033] Table 1 Primers used in the construction process

[0034]

[0035] Table 2. sgRNAs used in mutant construction

[0036]

[0037] Example 2: Obtaining rice with OsLTPL113 gene knockout of the rice lipid transporter

[0038] This invention utilizes CRISPR / Cas9 technology. First, a knockout vector for OsLTPL113 is constructed. Then, the sgRNA of the OsLTPL113 gene is designed using the CRISPR-P2.0 website (http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR). Finally, using the same method as described above, a CRISPR / Cas9 knockout vector carrying the OsLTPL113 sgRNA is constructed.

[0039] The knockout vector was then chemically transformed into Agrobacterium EHA105, and positive single colonies were selected to prepare OD. 600 Rice ZH11 callus was infected with a 0.2% inoculum solution for 15 min, followed by incubation at 20 ℃ for 48-72 h. Single-clone callus tissue was then selected and cultured on a selection medium at 26 ℃ in the dark. After 25 days, positive single-clone callus tissue was selected and subcultured at 26 ℃ in the dark. After 7-10 days of culture, the positive callus tissue was inoculated onto a differentiation medium and cultured at 27 ℃ under light for 15 days. Then, the differentiated buds (3-5 cm long) were inoculated onto a rooting medium and cultured at 30 ℃ under light for 7 days. 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.

[0040] After two breeding cycles, we obtained a knockout mutant of the OsLTPL113 gene. Sequencing confirmed that the gene had been knocked out and that two different mutations existed. Figure 1 A).

[0041] Example 3: Analysis of resistance and susceptibility between rice varieties overexpressing and knocking out the OsLTPL113 gene.

[0042] This invention propagates overexpressing and knockout transgenic rice plants to obtain third-generation stably inherited progeny plants for pathogenicity testing. By inoculating rice leaves with spores of the wild-type strain Guy11 of *Oryza sativa* and fungal cakes of the wild-type strain AG-1IA of *Rhizoctonia solani*, the pathogen of rice sheath blight, it was found that the OsLTPL113 mutant was susceptible to disease, while plants overexpressing the OsLTPL113 gene showed significant resistance to both *Oryza sativa* and *Rhizoctonia solani*. Figure 2 A, 2C); further measurements of the length or area of ​​lesions on rice leaves also significantly demonstrated the contribution of the OsLTPL113 gene to the resistance of rice to fungal diseases. Figure 2B、2D)。

[0043] SEQ ID NO:1

[0044] AGGAGTCTTTTGACAACAACAGATTGCTAGGGTTCTGGTTCTCTCCCCTACAAATACCCACGTACTCTGCTGAGTTTTCGCACCTCTCTCTAGTTGCAGACCATCACTTACGTAGCCCTGTGTGCAACGGCGCAAGTGCTTGTACGCTTTCAGCTAGCGTAGCCATGGCTTCCAGGGCATTCCTCCTCGTGGCTCTAAACCTGGTCCTCTTCTTCACCGTGGCCAGCGCCTGCGGCAAGTACTGCCCGACGCCTTCGACGCCGTCGACGACGCCATCGACGCCGTCCTACAACACCAAGTGCCCCAAGAACGCGCTCAAGTTCGCGGCGTGCGCCGACGTGCTGGGCCTCGTCAGCGCCGAGGTCGGCCAGCCGCCGTACGAGCCGTGCTGCGGCGTCCTCGGCGGCCTCGCCGACCTTGAGGCCGCCGTCTGTCTCTGCACCGCCATCAAGGCCAACGTGCTCGGCATCACCCTCGACATCCCCGTCAAGCTCAGCCTCCTCGTCAACTACTGCGGCAAGAACGTCCCTAGTGGCTTCATCTGTGCTTAAGCTACGTAACGCGCGTACGGTGTAACGACGTGCTAGCTTTGCATGCATGCAGCACGCATGCACGAACACATCGTTCGTTCTTGAGTGCCTGCATGCATATCGGTCGAGTCTTTACTTACTCTGTTATTAGTTCTGAATGTAGAACTGCTTCAGATATCAATCCAGCGAGTTAACTGTACTTGATTTGTTTATGTTTCTTCAGTTAATCAGTTTCTGTCATCATTTGGTCAATTAATGGAGGTTTTGTTCTCCAAGGAATTGCATGCAATGGATACTCTTTTAGATTGAATTTGGCGTGTCTATGTCTGGGTGTGGTTGTAATTTGCTTGCAGTACGTTCTCGTAGTTTAATTGTAAGCAAGCAATTGTACGACAAAATGTACTTAGGACAGTTAATTTGTTGGAAATTTCGGTGTGAATTATTTTTTAAAATTTACA

[0045] SEQ ID NO:2

[0046] ATGGCTTCAGGGCATTCCTCCTCGTGGCTCTAAACCTGGTCCTCTTCTTCACCGTGGCCAGCGCCTGCGGCAAGTACTGCCCGACGCCTTCGACGCCGTCGACGACGCCATCGACGCCGTCCTACAACACCAAGTGCCCCAAGAACGCGCTCAAGTTCGCGGCGTGCGCCGACGTGCTGGGCCTCGTCAGCG CCGAGGTCGGCCAGCCGCCGTACGAGCCGTGCTGCGGCGTCCTCGGCGGCCTCGCCGACCTTGAGGCCGCCGTCTGTCTCTGCACCGCCATCAAGGCCAACGTGCTCGGCATCACCCTCGACATCCCCGTCAAGCTCAGCCTCCTCGTCAACTGCGGCAAGAACGTCCCTAGTGGCTTCATCTGTGCTTAA

[0047] SEQ ID NO:3

[0048] MASRAFLLVALNLVLFFTVASACGKYCPTPSTPSTTPSTPSYNTKCPKNALKFAACADVLGLVSAEVGQPPYEPCCGVLGGLADLEAAVCLCTAIKANVLGITLDIPVKLSLLVNYCGKNVPSGFICA

[0049] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A rice lipid transporter protein, OsLTPL113, characterized in that, The protein is selected from any one of the following (A1)-(A3): (A1) A protein with the amino acid sequence shown in SEQ ID NO:3; (A2) Proteins derived from 1) with the same activity, but with one or more amino acids substituted, deleted, and / or added to the amino acid sequence shown in SEQ ID NO.3; (A3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of (A1) or (A2).

2. The rice lipid transporter OsLTPL113 gene, selected from any one of the following (A1)-(A3): (A1) The nucleotide sequence is shown in SEQ ID NO:1; (A2) The CDS sequence is shown in SEQ ID NO:2; (A3) A nucleotide sequence that hybridizes with a DNA sequence defined by (A2) under strict conditions.

3. A recombinant expression vector, expression cassette, transgenic cell line, or genetically engineered bacterium containing the rice lipid transporter OsLTPL113 gene as described in claim 2.

4. A method for improving the resistance of rice to fungal diseases, characterized in that, The rice lipid transporter OsLTPL113 gene of claim 2 is overexpressed in rice, wherein the rice lipid transporter OsLTPL113 gene is a gene encoding the protein with the amino acid sequence shown in SEQ ID NO:

3.

5. The method according to claim 4, characterized in that, The overexpression method is as follows: the rice lipid transporter OsLTPL113 gene is cloned and a plant expression vector is constructed. The plant expression vector is then transformed into Agrobacterium, and rice is inoculated. Overexpression lines or their progeny with fungal resistance are selected.

6. The method according to claim 4 or 5, characterized in that, The CDS sequence of the rice lipid transporter OsLTPL113 gene is shown in SEQ ID NO:

2.

7. The method according to claim 5, characterized in that, The plant expression vector is pXQ; Preferably, the Agrobacterium is EHA105.

8. The application of the rice lipid transporter OsLTPL113 as described in claim 1, the rice lipid transporter OsLTPL113 gene as described in claim 2, or the recombinant expression vector, expression cassette, transgenic cell line, or genetically engineered bacteria as described in claim 3 in rice resistance to fungal diseases or in the preparation of rice products resistant to fungal diseases.

9. The application of the rice lipid transporter OsLTPL113 of claim 1, the rice lipid transporter OsLTPL113 gene of claim 2, or the recombinant expression vector, expression cassette, transgenic cell line or genetically engineered bacteria of claim 3 in the cultivation of rice varieties with enhanced resistance to fungal diseases or in the preparation of products that cultivate rice varieties with enhanced resistance to fungal diseases.

10. The application of the rice lipid transporter OsLTPL113 of claim 1, the rice lipid transporter OsLTPL113 gene of claim 2, or the recombinant expression vector, expression cassette, transgenic cell line or genetically engineered bacteria of claim 3 in identifying rice varieties or plants with enhanced resistance to fungal diseases.

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