Use of a bacterial-derived tRNA restriction enzyme PrrC or its coding gene in improving plant disease resistance

Abstract

The application relates to application of a bacterial-derived tRNA restriction enzyme PrrC or a coding gene thereof in improving plant disease resistance, and belongs to the technical field of genetic engineering. The bacterial tRNA restriction enzyme is introduced into the field of plant disease resistance for the first time, and its function in plant disease resistance is verified, not only the plant disease resistance resource library is explored and expanded, it is proved that the bacterial immune-related proteins have a conservative immune function in plants, can assist the plant disease resistance, and meanwhile it is shown that a large number of immune-related genes still exist in bacteria and need to be further developed and utilized. The PrrC protein is heterologously expressed in plants, the tRNA restriction enzyme activity thereof is utilized to induce the hypersensitive necrosis reaction of the plant cells infected by a pathogenic substance, so that the expansion of the pathogenic substance is inhibited, and the plant disease resistance is improved.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the application of a bacterial tRNA restriction endonuclease PrrC or its encoding gene in enhancing plant disease resistance. Background Technology

[0002] Plant diseases pose a serious threat to food security and food safety, and enhancing plant disease resistance is one of the most economical and effective means of disease control. In recent years, significant breakthroughs have been made in the cloning of disease resistance genes, especially nucleotide-binding domain and leucine-rich repeat (NLR) receptors. However, because most disease resistance genes are specific in their recognition of pathogen effectors, they are difficult to meet the needs of agricultural production for broad-spectrum and durable disease resistance.

[0003] PrrC is the first bacterial tRNA restriction endonuclease discovered. It blocks bacteriophage transmission by specifically cleaving tRNA, leading to translation termination and cell death. However, whether this type of bacterial immune protein can exert immune functions across species in plants, and how to regulate its expression level in plants so that it can effectively initiate immune defense without affecting plant growth and development, remains unreported. Summary of the Invention

[0004] The purpose of this invention is to address the current situation where existing plant disease resistance genes are insufficient to meet the needs of agricultural production, and to provide a solution for cross-species application of bacterial tRNA restriction endonucleases in plant disease resistance breeding. To this end, this invention provides the application of the bacterial tRNA restriction endonuclease PrrC or its encoding gene in enhancing plant disease resistance.

[0005] This invention provides the application of a bacterial tRNA restriction endonuclease PrrC or its encoding gene in enhancing plant disease resistance, wherein the amino acid sequence of the tRNA restriction endonuclease PrrC is shown in SEQ ID NO.1.

[0006] Preferably, the application includes: enhancing plant disease resistance by expressing the tRNA restriction endonuclease PrrC in biological materials.

[0007] Preferably, the biological material includes a recombinant expression vector containing a gene encoding the tRNA restriction endonuclease PrrC, a recombinant host cell, or a transgenic plant cell.

[0008] Preferably, the gene encoding the tRNA restriction endonuclease PrrC is expressed by the MAS promoter.

[0009] Preferably, the gene encoding the tRNA restriction endonuclease PrrC is a plant codon-optimized gene, and the nucleotide sequence of the plant codon-optimized gene is shown in SEQ ID NO.3.

[0010] Preferably, the enhancement of plant disease resistance includes any one or more of the following ① to ④: ① Improve plant resistance to oomycete diseases; ② Improve plant resistance to viral diseases; ③ Upregulate the expression levels of plant disease resistance-related genes, including... PR1b , PR2b , PR4 and LOX At least one of them; ④ Induces plants to produce an allergic necrosis response.

[0011] Preferably, the oomycete includes Phytophthora capsici; the virus includes turnip mosaic virus.

[0012] This invention also provides a method for improving plant disease resistance, comprising the following steps: The gene encoding the PrrC protein was introduced into plants to enable the plants to heterologously express the PrrC protein. The amino acid sequence of the PrrC protein is shown in SEQ ID NO.1.

[0013] This invention also provides a method for cultivating highly disease-resistant plants, comprising the following steps: Transgenic plants obtained through the methods described in the above technical solution are used as parents for breeding.

[0014] The present invention also provides a plant material with high disease resistance, wherein the plant material heterologously expresses the PrrC protein; the amino acid sequence of the PrrC protein is shown in SEQ ID NO.1.

[0015] Beneficial effects: This invention provides an application of bacterial tRNA restriction endonuclease PrrC or its encoding gene in enhancing plant disease resistance. By heterologously expressing the PrrC protein in plants, its tRNA restriction endonuclease activity induces a hypersensitive necrosis response in pathogen-infected plant cells, thereby inhibiting the spread of pathogens and enhancing plant disease resistance.

[0016] This invention is the first to introduce bacterial tRNA restriction endonucleases into the field of plant disease resistance and confirm their function in plant disease resistance. It not only explores and expands the plant disease resistance resource library, but also proves that bacterial immune-related proteins have conserved immune functions in plants and can assist plants in disease resistance. At the same time, it also shows that there are still a large number of immune-related genes in bacteria that need to be further developed and utilized.

[0017] Building upon this foundation, the present invention also proposes a flexible and efficient new strategy for plant disease resistance breeding. This involves selecting suitable plant promoters and recombinantly expressing resistance genes to confer resistance to different pathogens in plants. Verification through examples shows that by selecting appropriate promoters (MAS promoter, Mannopine synthase promoter) to drive the PrrC encoding gene, and combining this with plant codon optimization, PrrC can be successfully expressed in plants through cross-species recombination, conferring broad-spectrum resistance to various pathogens such as oomycetes and viruses, without affecting normal plant growth and development. Attached Figure Description To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 The pSuper1300 full-sequence GFP FLAG vector map; Figure 2 The graph shows the functional phenotype and biochemical detection results of the PrrC mutant; among them... This indicates a p-value < 0.001; Figure 3 The image shows the results of the relative gene expression level detection in the PrrC transgenic line; among them... represent P Value < 0.0001; Figure 4 The image shows the resistance phenotype of PrrC transgenic tobacco to Phytophthora capsici and the quantitative detection results of the pathogen; among them, represent P Value < 0.0001; Figure 5Figure 1 shows the inhibition phenotype of TuMV systemic infection in PrrC transgenic tobacco and the results of quantitative virus detection. Figure A shows the detection results of TuMV-GFP fluorescence infection phenotype in whole leaves of different strains under ultraviolet fluorescence (n=9); Figure B shows close-up images of ultraviolet fluorescence infection in individual leaves of each strain; and Figure C shows the detection results of relative TuMV viral biomass. represent P Value < 0.0001; Figure 6 This figure shows the comparison of growth, development, and agronomic traits between PrrC transgenic tobacco and wild-type Nicotiana benthamiana. Detailed Implementation

[0019] This invention provides the application of a bacterial tRNA restriction endonuclease PrrC or its encoding gene in enhancing plant disease resistance, wherein the amino acid sequence of the tRNA restriction endonuclease PrrC is shown in SEQ ID NO.1. SEQ ID of the present invention NO.1 is specifically: MGKTLSEIAQQLSTPQKVKKTVHKEVEATRAVPKVQLIYAFNGTGKTRLSRDFKQLLESKVHDGEGEDEAEQSALSRKKILYYNAFTEDLFYW DNDLQEDAEPKLKVQPNSYTNWLLTLLKDLGQDSNIVRYFQRYANDKLTPHFNPDFTEITFSMERGNDERSAHIKLSKGEESNFIWSVFYTLLDQVVTILN VADPDARETHAFDQLKYVFIDDPVSSLDDNHLIELAVNLAGLIKSSESDLKFIITTHSPIFYNVLFNELNGKVCYMLESFEDGTFALTEKYGDSNKSFSYHLHLKQTIEQAIADNNVERYHFTLLRNLYEKTASFLGYPKWSELLPDDKQLYLSRIINFTSHSTLSNEAVAEPTPAEKATVKLLLDHLKNNCGFWQQEQKNG.

[0020]

[0021] In one implementation method, this invention heterologously expresses the PrrC protein in plants, utilizing its tRNA restriction endonuclease activity to induce a hypersensitive necrosis response in pathogen-infected plant cells, thereby inhibiting pathogen spread and enhancing plant disease resistance. This invention is the first to introduce bacterial tRNA restriction endonucleases into the field of plant disease resistance and confirm their function in plant disease resistance. It not only explores and expands the plant disease resistance resource pool and demonstrates that bacterial immune-related proteins possess conserved immune functions in plants, assisting in plant disease resistance, but also indicates that a large number of immune-related genes in bacteria remain to be further developed and utilized.

[0022] As one implementation, the application of the present invention includes: enhancing plant disease resistance by expressing the tRNA restriction endonuclease PrrC in biological materials.

[0023] In one embodiment, the biological material of the present invention includes a recombinant expression vector containing the gene encoding the tRNA restriction endonuclease PrrC, recombinant host cells, or transgenic plant cells. In one embodiment, the initial vector of the recombinant expression vector of the present invention is the pCAMBIAsuper1300 vector. In one embodiment, the gene encoding the tRNA restriction endonuclease PrrC of the present invention is inserted into the multiple cloning site of the vector via homologous recombination. In one embodiment, the gene encoding the tRNA restriction endonuclease PrrC of the present invention includes an N-terminal NTPase domain and a C-terminal anticodon nuclease domain, both of which are essential for PrrC to perform its tRNA restriction endonuclease function. The N-terminal NTPase domain includes the R48 key site, and the C-terminal anticodon nuclease domain includes the R320, R349, and H356 key sites.

[0024] In one embodiment, the gene encoding the tRNA restriction endonuclease PrrC described in this invention is expressed by a MAS promoter. In another embodiment, the MAS promoter described in this invention was determined through screening various plant promoters. This MAS promoter can drive the expression level of the PrrC protein in plants to achieve a balance between effectively initiating immune defense and not affecting normal plant growth and development. The nucleotide sequence of the MAS promoter described in this invention is shown in SEQ ID NO. 30. SEQ ID of the present invention NO.30 is specifically: TTTTCAAATCAGTGCGCAAGACGTGACGTAAGTATCCGAGTCAGTTTTTATTTTTCTACTAATTTGGTCGTTTATTTCGGCGTGTAGGACATGGCAACCGGGCCTGAATTTCGCGGGTATTCTGTTTCTATTCCAACTTTTTCTTGATCCGCAGCCATTAACGACTTTTGAATAGATACGCTGACA CGCCAAGCCTCGCTAGTCAAAAGTGTACCAAACAACGCTTTACAGCAAGAACGGAATGCGCGTGACGCTCGCGGTGACGCCATTTCGCCTTTTCAGAAATGGATAAATAGCCTTGCTTCCTATTATATCTTCCCAAATTACCAATACATTACACTAGCATCTGAATTTCATAACCAATCTCGATACACCAAATCG.

[0025] In one implementation method, the present invention uses the MAS promoter to drive the PrrC encoding gene and combines it with plant codon optimization to successfully express PrrC across species in plants, thereby conferring broad-spectrum resistance to different pathogens such as oomycetes and viruses, without affecting the normal growth and development of plants.

[0026]

[0027] In one embodiment, the present invention inserts an exogenous IV2 intron into the optimized coding sequence, the sequence of which is shown in SEQ ID NO. 31. Specifically, SEQ ID NO. 31 is: GTAAGTTTCTGCTTCTACCTTTGATATATATATAATAATTATCATTAATTAGTAGTAATATAATATTTCAAATATTTTTTTCAAAATAAAAGAATGTAGTATATAGCAATTGCTTTTCTGTAGTTTATAAGTGTGTATATTTTAATTTATAACTTTTCTAATATATGACCAAAATTTGTTGATGTGCAG. In another embodiment, the exogenous IV2 intron cannot be spliced ​​in prokaryotes, thereby blocking the translation of PrrC protein in prokaryotic hosts such as Escherichia coli and preventing its cytotoxicity to the host bacteria; the intron can be normally spliced ​​out in plants without affecting the correct expression of PrrC protein in plants.

[0028] As one implementation method, the improvement of plant disease resistance according to the present invention includes any one or more of the following ① to ④: ① Improve plant resistance to oomycete diseases; ② Improve plant resistance to viral diseases; ③ Upregulate the expression levels of plant disease resistance-related genes, including... PR1b , PR2b , PR4 and LOX At least one of them; ④ Induces plants to produce an allergic necrosis response.

[0029] In one implementation method, the enhancement of plant disease resistance described in this invention does not significantly affect the normal growth and development of the plant.

[0030] In one embodiment, the oomycetes described in this invention include *Phytophthora capsici* (…). Phytophthora capsici The viruses mentioned include turnip mosaic virus (TuMV).

[0031] This invention also provides a method for improving plant disease resistance, comprising the following steps: The gene encoding the PrrC protein was introduced into plants to enable the plants to heterologously express the PrrC protein. The amino acid sequence of the PrrC protein is shown in SEQ ID NO.1.

[0032] In one embodiment, the present invention achieves the introduction through Agrobacterium-mediated transformation. In one embodiment, the plant described in the present invention is tobacco. In one embodiment, the transgenic plant obtained after the introduction of the present invention shows improved disease resistance, while its seed germination rate, maximum leaf area, and plant height are not significantly different from the wild type.

[0033] This invention also provides a method for cultivating highly disease-resistant plants, comprising the following steps: Transgenic plants obtained through the methods described in the above technical solution are used as parents for breeding.

[0034] As one implementation method, the application of the present invention introduces bacterial tRNA restriction endonuclease PrrC into plants through transgenic technology, which significantly shortens the disease resistance breeding cycle and avoids the directional selection pressure exerted by traditional disease-resistant varieties on field pathogens (such as resistance being overcome and rendered ineffective by new pathogen races after a few years), thus extending the effective use period of the disease resistance gene.

[0035] This invention also provides a plant material with high disease resistance, wherein the plant material heterologously expresses the PrrC protein; the amino acid sequence of the PrrC protein is shown in SEQ ID NO.1. As one embodiment, the plant material of this invention includes plants, seeds, tissues, and callus tissue.

[0036] To further illustrate the present invention, the application of a bacterial tRNA restriction endonuclease PrrC or its encoding gene in enhancing plant disease resistance is described in detail below with reference to the accompanying drawings and embodiments. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1 Codon optimization of bacterial PrrC encoding genes The bacterial PrrC protein sequence (NCBI sequence number: WP_032251799.1) was obtained from the NCBI database, and its amino acid sequence is shown in SEQ ID NO.1. The protein sequence was codon optimized using an online codon optimization tool (https: / / www.novopro.cn / tools / codon-optimization.html) targeting the rice codon. The original PrrC coding sequence before optimization is shown in SEQ ID NO.2. The plant codon-optimized PrrC coding gene was obtained, and its nucleotide sequence is shown in SEQ ID NO.3.

[0038] Furthermore, because the PrrC protein is cytotoxic to prokaryotes, a plant intron (IV2 intron) is inserted into the optimized PrrC coding sequence to facilitate gene cloning in *E. coli*, thereby blocking the translation of the PrrC protein in prokaryotes and preventing its toxicity to the host bacteria. This intron can be normally spliced ​​out in plants and does not affect the correct expression of the PrrC protein in plants.

[0039] Because the rice transgenic cycle is relatively long, the inventors first verified the expression effect of the optimized sequence in tobacco. The results showed that the PrrC coding sequence optimized for rice could also be expressed normally and function normally in tobacco.

[0040] Example 2 Transient expression of PrrC in tobacco induces cell necrosis and its functional verification. 1. Carrier Construction Allele fragments of bacterial PrrC and its key site mutations were amplified and constructed into the pCAMBIAsuper1300 vector via homologous recombination. The recombinant vector map is shown below. Figure 1 As shown.

[0041] The primer sequences are designed as follows: P1300-PrrC-GFP-F (SEQ ID NO.4): 5'-CCAAATCGACTCTAGAAAGCTTATGGGCAAGACTCTGTCTGAG-3'; P1300-PrrC-GFP-R (SEQ ID NO.5): 5'-TTGCTCACCATGGTCTCTCCGTTCTTCTGTTCTTGCT-3'; P1300-PrrC R48A -R (SEQ ID NO.6): gagaagctgcttgaaaTCCCTAGAAAGTGCGGTCTTGC; P1300-PrrC R48A -F (SEQ ID NO.7): gggaTTTCAAGCAGCTTCGAAAGC; P1300-PrrC R320A -R (SEQ ID NO.8): gcggtcttctcGTACAAGTTTGCGAGCAGCGT; P1300-PrrC R320A -F (SEQ ID NO.9): aacttgtacGAGAAGACCGCCTCGTTTCTC; P1300-PrrC R349A -R (SEQ ID NO.10): gagtgctgtgagacgtgaaATTGATGATTGCGGAGAGGTACA; P1300-PrrC R349A -F (SEQ ID NO.11): tTTCACGTCTCACAGCACTCTCAG; P1300-PrrC H356A -R (SEQ ID NO.12): cttcgttGCTGAGAGGTGCTTGCAGACGT; P1300-PrrC H356A -F (SEQ ID NO. 13): aagcactctcagcAACGAAGCGGTCGCTGAGC.

[0042] Amplification of PrrC and its key mutation allele fragments: Primer pairing was performed using P1300-PrrC-GFP-F and P1300-PrrC-GFP-R to amplify the wild-type PrrC fragment; each allele fragment was split into two fragments from the mutation site and amplified separately, with P1300-PrrC-GFP-F amplified with P1300-PrrC-GFP-R. R48A -R、P1300-PrrC R320A -R、P1300-PrrC R349A -R and P1300-PrrC H356A -R pairing is used to synthesize the portion preceding the mutation site, P1300-PrrC-GFP-R and P1300-PrrC... R48A -F、P1300-PrrC R320A -F、P1300-PrrC R349A -F and P1300-PrrC H356A -F pairing was used to synthesize the portion following the mutation site, and the synthesized PrrC fragment was used as a template for PCR amplification. The reaction conditions were: 95℃ pre-denaturation for 5 min; 95℃ for 30 sec, 58℃ for 30 sec, 72℃ for 30 sec, 34 cycles; 72℃ extension for 5 min.

[0043] Homologous recombination ligation: The pCAMBIAsuper1300 vector was digested with Hind III. Using a multi-fragment homologous recombination reagent (2×Seamless Cloning Mix, Boyuan Biotechnology, #RDA01), the above PCR fragments (wild-type PrrC fragment directly ligated; upstream and downstream fragments of each mutant ligated with the linearized vector via multi-fragment homologous recombination) were ligated to the linearized vector to construct transient expression vectors for each tobacco species. These vectors were then transformed into E. coli DH5α to obtain vectors containing allele fragments of PrrC and its key mutation sites. Expression of each vector was driven by the MAS promoter inherent in the pCAMBIAsuper1300 vector.

[0044] 2. Immune Characterization Transient expression of PrrC and its mutant alleles in Nicotiana benthamiana induces a plant hypersensitivity (HR) response: The experiment consisted of six groups: PrrC, PrrC alleles (R48A, R320A, R349A, and H356A), and the pCAMBIAsuper1300 empty vector (EV). R48A is located in the N-terminal NTPase domain, while R320A, R349A, and H356A are located in the C-terminal anticodon nuclease domain. Mutations at key sites in these two domains affected the restriction endonuclease function of PrrC. Each vector was transformed into Agrobacterium GV3101, and after 16 hours of shaking culture, the cells were resuspended in infection solution (10 mM MgCl2, 10 mM MES, and 100 μM acetylsylcholine), and the OD values ​​of each component were adjusted. 600 Let the solution stand for at least 3 hours. Inject each bacterial solution into four-week-old *Nicotiana benthamiana* leaves. 72 hours after injection, observe and photograph the tobacco leaves under white light and ultraviolet (UV) light. The results are as follows: Figure 2 The results show that PrrC can induce a human hazard response (HR) in tobacco cells. However, the PrrC allele, due to a mutation at a key site that inhibits tRNA restriction endonuclease activity, remains consistent with the empty vector (EV) and does not induce an HR response in tobacco cells.

[0045] Expression of PrrC and its mutant alleles significantly increases the ion leakage rate of tobacco cells: Following the same method described above, bacterial suspensions from each treatment group were injected into four-week-old *Nicotiana benthamiana* leaves. 24 hours after injection, 10 leaf discs (3 mm in diameter) from each treatment group were collected and equilibrated in 5 mL of sterile ddH2O for 1 hour. The conductivity of the samples was measured using a conductivity meter (FiveGo F3, Mettler-Toledo). The initial conductivity value was recorded as A. Care was taken not to damage the leaves during the measurement. After measurement, the test tubes containing the samples were transferred to boiling water and boiled for 30 minutes. After cooling, the conductivity of the leaves was measured again using the conductivity meter. The conductivity value after boiling was recorded as B. The ion leakage rate was calculated using the formula (A / B) × 100%. At least five biological replicates were performed for each treatment (Table 1). The ion leakage rate results are shown below. Figure 2 As shown, the ion leakage rate of PrrC, which can induce tobacco cell necrosis, is significantly higher than that of the PrrC mutant and EV, which cannot induce cell necrosis.

[0046] Table 1 Ion leakage rate results

[0047] The above results indicate that the bacterial PrrC protein can function as a conserved tRNA restriction endonuclease in plants, inducing a typical hypersensitive necrosis response. This function depends on the integrity of its N-terminal NTPase domain (R48 site) and C-terminal anticodon nuclease domains (R320, R349, and H356 sites). Mutations at any of the key sites in the PrrC alleles failed to induce tobacco cell necrosis, further confirming that PrrC-induced cell death in plants depends on its tRNA restriction endonuclease activity.

[0048] Example 3 Analysis of the resistance of PrrC transgenic tobacco to oomycete Phytophthora capsici and virus TuMV 1. Obtaining PrrC transgenic tobacco lines The bacterial PrrC gene fragment was amplified and constructed into the pCAMBIAsuper1300 vector through homologous recombination.

[0049] P1300-PrrC-F (SEQ ID NO. 14): 5'-CCAAATCGACTCTAGAAAGCTTATGGGCAAGACTCTGTCTGAG-3'; P1300-PrrC-R (SEQ ID NO. 15): 5'-TTGCTCACCATGGTCTCTCATCCGTTCTTCTGTTCTTGCT-3'.

[0050] The constructed vector was used to transform PrrC transgenic tobacco lines with Agrobacterium, and two independent lines were screened for subsequent experiments.

[0051] 2. Detection of expression levels of disease resistance-related genes in PrrC transgenic tobacco The experiment was divided into three groups, collecting young leaf tissues from wild-type Nicotiana benthamiana and independent lines M3 and M10 of PrrC transgenic Nicotiana, respectively, and rapidly freezing them in liquid nitrogen. The tissues were then ground in liquid nitrogen for RNA extraction.

[0052] Total RNA was extracted from the samples using the RNAprep Pure Plant Kit (TianGen Biotech; China). RNA was purified from the total RNA using DNase I (Thermo Scientific). Complementary DNA (cDNA) was synthesized using the HiScript II First-Strand cDNA Synthesis Kit (Vazyme). qRT-PCR was performed using SYBR Green Mixture (Vazyme) on a Bio-Rad CFX96 real-time system and a C1000 thermal cycler (Bio-Rad). Two [units / items / etc.] were used. -ΔΔCT The method calculates the relative expression levels of each gene. The primer sequences used are as follows: qRT-NbEF1α-F (SEQ ID NO. 16): agaggccctcagacaaac; qRT-NbEF1α-R (SEQ ID NO. 17): taggtccaaaggtcacaa; qRT-NbPR1b-F (SEQ ID NO. 18): gtggacactatactcaggtg; qRT-NbPR1b-R (SEQ ID NO. 19): tccaacttggaatcaaaggg; qRT-NbPR2b-F (SEQ ID NO. 20): aggtgtttgctatggaatgc; qRT-NbPR2b-R (SEQ ID NO. 21): ctgtaccccaccatcttgc; qRT-NbPR4-F (SEQ ID NO. 22): ggccaagattcctgtggtagat; qRT-NbPR4-R (SEQ ID NO. 23): cactgttgtttgagttcctgttcct; qRT-NbLOX-F (SEQ ID NO. 24): aaaacctatgcctcaagaac; qRT-NbLOX-R (SEQ ID NO. 25):actgctgcataggctttgg.

[0053] The results showed that, compared with wild-type Nicotiana benthamiana, the expression levels of disease resistance-related genes such as PR1b, PR2b, PR4, and LOX were significantly upregulated in the PrrC transgenic lines M3 and M10 (Table 2). Figure 3 This indicates that the expression of PrrC activates the expression of plant disease resistance-related genes.

[0054] Table 2. Relative expression levels of each gene

[0055] 3. Analysis of the resistance of PrrC transgenic tobacco to oomycete Phytophthora capsici. When the transgenic tobacco plants reached four weeks of age, leaves from wild-type Nicotiana benthamiana (negative control) and PrrC transgenic tobacco lines M3 and M10 were cut and placed in inoculation trays lined with moistened absorbent paper towels. A 3 mm diameter mycelial cake of Phytophthora capsici strain LT263 was inoculated onto the leaf surface, and 10 μL of sterile ddH2O was added to the inoculation site. The trays were then covered with plastic wrap to maintain moisture and protected from light for 36–48 h. Subsequently, the leaves were observed and photographed under ultraviolet (UV) light, and the lesion area was statistically analyzed using ImageJ software. The results showed that the lesion area on the leaves of transgenic lines M3 and M10 was significantly smaller than that of the wild-type control group (Table 3). Figure 4 ).

[0056] Table 3. Area of ​​Phytophthora infestation in peppers

[0057] Leaf tissue samples with a diameter of 3 cm were excised from the inoculation site, and total DNA was extracted using the CTAB method. Using tobacco EF1α as an internal control gene and *Phytophthora capsici* Pc-tubulin as the pathogen detection gene, the relative biomass of *Phytophthora capsici* in the leaf tissue was determined by qRT-PCR. The primers used were: Pc-tubulin-F (SEQ ID NO. 26): CAGAGGGTGCTGAGCTTATTGACT; Pc-tubulin-R (SEQ ID NO. 27):GAGAGTGGGTGATCTGGAAACCC.

[0058] qRT-PCR was performed using SYBR Green mixed buffer (Vazyme) on a Bio-Rad CFX96 real-time system and a C1000 thermal cycler (Bio-Rad). Two... -ΔΔCT The method was used to calculate the relative biomass of Phytophthora capsici in tobacco leaf tissue.

[0059] The results showed that the relative biomass of Phytophthora capsici in the leaf tissues of the PrrC transgenic lines M3 and M10 was significantly lower than that in the wild-type control group (Table 4), further confirming that the introduction of PrrC enhanced the resistance of tobacco to Phytophthora capsici.

[0060] Table 4 Relative Pathogen Biomass

[0061] 4. Resistance analysis of PrrC transgenic tobacco to TuMV When the transgenic tobacco plants reached three weeks of age, two fully developed leaves of wild-type Nicotiana benthamiana (negative control) and PrrC transgenic tobacco lines M3 and M10 were injected with Agrobacterium tumefaciens (OD) containing the TuMV genome information. 600 =0.5) were inoculated. One week later, the infection status of TuMV on the leaves of the grafted plant systems was observed under ultraviolet light. It was found that the TuMV infection level in the leaves of the M3 and M10 grafted plant systems was significantly lower than that in the wild type ( Figure 5 ).

[0062] Equal volumes of systematic leaf tissue were collected, cryogenically ground in liquid nitrogen, and total RNA was extracted from the samples using the RNAprep Pure Plant Kit (TianGenBiotech; China). RNA was purified from the total RNA using DNase I (Thermo Scientific). Complementary DNA (cDNA) was synthesized using the HiScript II first-strand cDNA synthesis kit (Vazyme). Tobacco EF1α was used as an internal reference gene, along with the TuMV coat protein gene. TuMV-CP To detect the target, qRT-PCR was performed. The primer sequences for detecting the relative biomass of Phytophthora capsici in leaf tissue were as follows: qRT-TuMV-CP-F (SEQ ID NO. 28): CACGCCGGAGCAGACGGATC; qRT-TuMV-CP-R (SEQ ID NO. 29): CTGATCGTCGCCGTCCATCATC.

[0063] qRT-PCR was performed using SYBR Green mixed buffer (Vazyme) on a Bio-Rad CFX96 real-time system and a C1000 thermal cycler (Bio-Rad). Two...-ΔΔCT The method was used to calculate the relative biomass of TuMV in tobacco leaf tissues.

[0064] The results showed that the relative biomass of TuMV in the leaves of the M3 and M10 systems was significantly lower than that in the wild-type control group. Simultaneously, total protein was extracted from equal amounts of leaf tissue from the systems and detected by Western blotting using an anti-GFP antibody (HUABio GFP Recombinant Rabbit Monoclonal Antibody [SP069-0], catalog number ET1604-26, dilution ratio 1:5000). The results showed that the expression level of TuMV-GFP protein in the leaves of the M3 and M10 systems was also significantly lower than that in the wild-type control group, further confirming that PrrC transgenic tobacco has enhanced resistance to TuMV.

[0065] Example 4 Growth phenotypes and agronomic traits of PrrC transgenic tobacco To verify the effect of PrrC transfer on tobacco growth and development, the following growth indicators of wild-type Nicotiana benthamiana and PrrC transgenic lines M3 and M10 were compared and analyzed.

[0066] 1. Seed germination rate Thirty seeds each of the plump and healthy transgenic lines M3 and M10, and wild-type Nicotiana benthamiana, harvested at the same time, were sown on 1 / 2 MS medium and placed at 25°C, 60% humidity, and 12h light (80 μmol / m²). -2 s -1 Seeds were germinated in the dark for 12 hours, and germination was defined as the emergence of two cotyledons. Germination rate was calculated after 6 days, and the results showed no significant difference in seed germination rate among the three lines.

[0067] 2. Maximum leaf area When the tobacco plants reached four weeks of age, the leaf area of ​​the largest fully expanded leaves of the wild-type and transgenic lines M3 and M10 was measured (the largest fully expanded leaves of each line were selected and measured using ImageJ software). The results showed no significant difference in the maximum leaf area among the three lines.

[0068] 3. Plant height When the tobacco plants reached eight weeks of age, the plant height (from soil surface to top) of the wild-type and transgenic lines M3 and M10 was measured. The results showed no significant difference in plant height among the three lines.

[0069] The above results indicate that the introduction of PrrC had no significant effect on seed germination, leaf growth, and plant height in tobacco, meaning that PrrC enhanced plant disease resistance without affecting normal plant growth and development. Figure 6 ).

[0070] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of a bacterial tRNA restriction endonuclease PrrC or its encoding gene in enhancing plant disease resistance, wherein the amino acid sequence of the tRNA restriction endonuclease PrrC is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The applications include: enhancing plant disease resistance in biological materials expressing the tRNA restriction endonuclease PrrC.

3. The application according to claim 2, characterized in that, The biological materials include recombinant expression vectors containing the gene encoding the tRNA restriction endonuclease PrrC, recombinant host cells, or transgenic plant cells.

4. The application according to claim 3, characterized in that, The gene encoding the tRNA restriction endonuclease PrrC is expressed by the MAS promoter.

5. The application according to claim 4, characterized in that, The gene encoding the tRNA restriction endonuclease PrrC is a plant codon-optimized gene, and the nucleotide sequence of the plant codon-optimized gene is shown in SEQ ID NO.

3.

6. The application according to any one of claims 1 to 5, characterized in that, The enhancement of plant disease resistance includes any one or more of the following ① to ④: ① Improve plant resistance to oomycete diseases; ② Improve plant resistance to viral diseases; ③ Upregulate the expression levels of plant disease resistance-related genes, including... PR1b , PR2b , PR4 and LOX At least one of them; ④ Induces plants to produce an allergic necrosis response.

7. The application according to claim 6, characterized in that, The oomycetes include Phytophthora capsici; the viruses include turnip mosaic virus.

8. A method for improving plant disease resistance, characterized in that, Includes the following steps: The gene encoding the PrrC protein was introduced into plants to enable the plants to heterologously express the PrrC protein. The amino acid sequence of the PrrC protein is shown in SEQ ID NO.

1.

9. A method for cultivating highly disease-resistant plants, characterized in that, Includes the following steps: The transgenic plant obtained by the method described in claim 8 is used as a parent for breeding.

10. A highly disease-resistant plant material, characterized in that, The plant material heterologously expresses the PrrC protein; the amino acid sequence of the PrrC protein is shown in SEQ ID NO.1.

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