Application of the reigen gene OSCBL1 and the protein encoded by it to increase resistance to bacterial leaf rot

Description

2 Calcium signals act as central second messengers in the plant response to biotic stress; the calcium sensor proteins include the calcineurin B-like subunit proteins (CBL), which regulate diverse physiological processes via the CBL-CIPK signaling network. In the state of the art,5 proteomic studies have shown that the expression level of OsCBL1 changes significantly in the context of the Xa21-mediated resistance reaction of rice against bacterial leaf rot. From this, it was deduced that OsCBL1 could be involved in the interaction between plant and pathogen (LiuYumeng, 10 LanJinping, CaoYinghaou a.: Expression of calcineurin B-like subunit proteins of rice during leaf development). Resistance reaction against bacterial leaf blight, ActaBotanica Sinica, 2012, Vol. 47, No. 5, pp. 483-490).Nevertheless, the aforementioned study has the following limitations: (1) The study only observed reduced protein expression of OsCBL1 in Xa21-carrying resistant rice varieties; validation on susceptible varieties was not performed; (2) The study was limited to protein-related detection using proteomics methods; functional testing of OsCBL1 by genetic techniques such as gene knockout or overexpression was not performed; (3) The study does not address the molecular mechanism by which OsCBL1 regulates rice resistance to bacterial leaf rot; (4) No specific technical implementation routes for disease-resistant breeding were provided, including no target sequences for genome editing, no vector construction methods, and no breeding procedures.For the aforementioned reasons, the experimental confirmation of the biological function of OsCBL1 in the regulation of resistance to bacterial leaf rot, the elucidation of the signaling mechanism mediating the outbreak of reactive oxygen species (ROS), and the generation of novel highly resistant breeding material are of practical importance for overcoming problems such as scarce resistance resources and rapid loss of resistance in brown rice varieties. 5 CONTENT OF THE INVENTION To overcome the disadvantages of the prior art, the present invention provides the use of the rice gene OsCBL1 and the protein encoded by it for the regulation of resistance to bacterial leaf rot in rice. The technical solution of the present invention is as follows: In the first aspect, the present invention provides the application of the OsCBL1 protein or the gene encoded by it to increase the resistance of rice against bacterial leaf rot.The 15 increase in rice resistance to bacterial leaf rot is achieved by reducing the expression level of the OsCBL1 gene and / or impairing the function of the OsCBL1 protein. Furthermore, the amino acid sequence of the OsCBL1 protein corresponds to SEQIDNO:2 and the nucleotide sequence of the OsCBL1 gene corresponds to SEQIDNO:1. Furthermore, the method for reducing the expression level of the OsCBL1 gene and / or impairing the function of the OsCBL1 protein represents a gene knockout. Preferably, the gene knockout method is based on genome editing using CRISPR / Cas9. Preferably, the target sequence of the sgRNA used in genome editing by means of CRISPR / Cas9 corresponds to SEQIDNO: 3. The target sequence is located in the coding region of the OsCBL1 gene and is suitable to effectively impair the biological function of the OsCBL1 protein. 30 BE2026 / 7372 4 In the second aspect, the present invention provides a CRISPR / Cas9 knockout vector for increasing the resistance of rice to bacterial leaf rot.The vector contains a γRNA targeting the OsCBL1 gene, wherein the target sequence of the γRNA corresponds to SEQIDNO:3.5 In the third aspect, the present invention provides a method for breeding rice resistant to bacterial leaf rot. The method comprises the following steps: (1) construction of a knockout vector for the OsCBL1 gene; (2) introduction of the vector constructed in step (1) into cells or tissue of the rice; (3) selection of rice plants with reduced expression levels of the OsCBL1 gene or with non-functional OsCBL1 protein; (4) testing the resistance of the plants obtained in step (3) to bacterial leaf rot. Preferably, the knockout vector according to step (1) is the CRISPR / Cas9 knockout vector according to the second aspect of the present invention. Preferably, the introduction according to step (2) is carried out by means of genetic transformation mediated by 20 Agrobacteria.In the fourth aspect, the present invention provides a rice plant resistant to bacterial leaf rot, which is obtainable according to the aforementioned method, as well as offspring, seeds, plant tissue, or plant cells.25 Compared to the prior art, the present invention has the following advantageous effects: (1) Clarification of the regulatory gene function: Through reverse and forward genetic experiments, it is demonstrated that OsCBL1 is a negative regulatory factor for the resistance of rice to bacterial leaf rot.30 A mere assumption regarding the relationship of protein expression is further developed into a result supported by functional testing, thereby supplementing the knowledge on the function of this gene under biotic stress. (2) Elucidation of the molecular mechanism of disease resistance:5 It is demonstrated that OsCBL1 influences rice susceptibility to bacterial leaf rot via the regulation of the accumulation of reactive oxygen species.A gene knockout of this gene promotes the release of superoxide anions after inoculation with the pathogen and provides an experimental basis for the regulation of plant immunity by calcium signaling. (3) Provision of a practical breeding target: The SEQIDNO:3 is confirmed as an effective target sequence for genome editing using CRISPR / Cas9, targeting the OsCBL1 gene. The resulting gene-knockout plants show significantly improved resistance to bacterial leaf rot and can be used directly for disease-resistant rice breeding. (4) Optimized advantages of the breeding application: Resistance is increased by modifying an endogenous rice gene without introducing foreign genes. This reduces disadvantages of the state of the art, such as a narrow resistance spectrum and rapid loss of disease resistance; the method is characterized by high biological safety and is suitable for large-scale breeding applications.25 DESCRIPTION OF THE ATTACHED DRAWINGS Figure 1 shows the expression pattern of the OsCBL1 gene after induction by the pathogen PXO99 in bacterial leaf rot of both susceptible rice varieties Nipponbare; *** represents P<0.001, **** represents P<0.0001, ns represents a statistically non-significant difference. BE2026 / 7372 6 Figure 2 shows the molecular identification of plants with gene knockout of the OsCBL1 gene: Part A shows the gRNA target sequence of the OsCBL1 gene as well as a sequence comparison of the target sites of wild type (WT) and knockout lines (KO1, KO2); Part B includes the electrophoresis of the PCR detection of the hygromycin resistance gene in the overexpression lines (OE1, OE2) as well as the relative expression level of the OsCBL1 gene.Figure 3 contains the investigation of the disease resistance of OsCBL1-transgenic rice after inoculation with the pathogen PXO99 for bacterial leaf rot: Part A is a bar chart for the statistical evaluation of the lesion lengths of different materials 14 days after inoculation; Part B shows the disease pattern of the rice leaves after 14-day inoculation; * means P<0.05, ** means P<0.01, **** means P<0.0001. Figure 4 shows the result of histochemical staining with NBT15 on OsCBL1-transgenic rice before and after inoculation with the pathogen PXO99 of bacterial leaf rot: Part A represents the staining result at 0 hours after inoculation; Part B the result after 24 hours of inoculation; Part C the result after 48 hours of inoculation. 20 DETAILED DESCRIPTION The present invention will be explained in more detail below with reference to exemplary embodiments, without limiting the invention to these exemplary embodiments.Unless otherwise stated, the experimental procedures used in the following examples are standard methods. The materials, reagents, etc. used below are, unless otherwise stated, commercially available. BE2026 / 7372 7 Experimental materials Rice variety: Nipponbare (Oryzasativa L. cv. Nipponbare) Pathogen: Isolate PXO99, bacterial leaf rot from the Philippines Transgenic rice plants with gene knockout of OsCBL1: KO1, KO-25 (homozygous lines of the T2 generation) Transgenic rice plants with overexpression of OsCBL1: OE-1, OE2 (homozygous lines of the T2 generation) Example 1: Analysis of the pathogen-induced expression of the OsCBL1 gene in the susceptible rice variety 10 1.1 Cultivation of the starting material A selection of plump rice seeds was made, the surface of which was first treated with 75% ethanol for a period of 50 seconds disinfection is followed by a single rinse with sterile water.The seeds are then disinfected for 30 minutes using a sodium hypochlorite solution with an effective chlorine content of 2.5%, followed by eight rinses with sterile water. The cleaned seeds are laid out on sterile filter paper to dry and then placed in an incubator at a constant temperature of 28°C for germination. Once the white tips of the seeds appear, they are sown in plastic pots filled with nutrient medium and cultivated in a climate chamber under the following conditions: daytime temperature 28°C, nighttime temperature 25°C, 16 hours of light per day, 8 hours of darkness per day, and a relative humidity of 70%. Inoculation is carried out after 25 months of rice cultivation. 1.2 Treatment with the pathogen Inoculation with the pathogen of bacterial leaf rot is carried out using the live leaf cut inoculation method. The activated Xoo strain PXO99A is resuspended with sterile water and adjusted to a 30 OD600 value of 0.5.Using sterile scissors moistened with the bacterial suspension BE2026 / 7372 8, approximately 2 to 3 cm of the tip of the rice leaves are cut off. The leaf samples are taken at 0 h, 1 h, 3 h, 6 h, 12 h, and 24 h after inoculation. The samples are preserved by freezing in liquid nitrogen and then stored at −80°C for later use. 1.3 Extraction of Total RNA and Synthesis of cDNA Total RNA is extracted from the leaves of rice sprouts using the TransZol reagent (TransGenBiotech, Beijing). The integrity of the extracted RNA is checked by electrophoresis in a 1.5% agarose gel. The synthesis of the first cDNA strand takes place using the PrimeScript™ Reagent Kit with DNA Eraser (TaKaRa, Dalian) according to the kit's instructions. The concentration of the synthesized cDNA is determined with a micro-spectrophotometer, which is then adjusted to a suitable concentration for subsequent quantitative experiments.4. Detection by quantitative real-time PCR. The quantitative PCR is performed according to the SYBR Green I method, using the rice gene ubiquitin 5 (UBQ5) as a reference gene. The detection of the expression level of the OsCBL1 gene 20 is carried out using the primers OsCBL1-qF / qR. Primer sequences for quantitative real-time PCR: SEQIDNO:4:OsCBL1-qF(5'-AGTGAGGTGGAGGCTTTGTT-3') SEQIDNO:5:OsCBL1-qR(5'-AGTCACCAAAGTCGATGACCC-3') SEQIDNO:6:ubiquitin5-F(5'-GCTCCGGTGGCGGTATCAT-3')25 SEQIDNO:7:ubiquitin5-R(5'-CGGCAGTTGACAGCCCTAG-3') Composition of the reaction mixture for quantitative PCR (20 μL): 10 μL 2×SYBRPremixExTaqII(TliRNaseHPlus), 0.8 μL each of forward and reverse primer (10 μmol / L), 2 μL cDNA template,30 Supplementation with sterile dH2O to a total volume of 20 μL. BE2026 / 7372 9 Reaction conditions of quantitative PCR: 30 s pre-denaturation at 95°C; cyclic execution of 5 s denaturation at 95°C and 34 s annealing at 60°C with detection of the fluorescence signal over a total of 40 cycles.The calculation of the relative expression values ​​is performed using the 2⁻ΔΔCt method.5 1.5 Results The evaluation of the quantitative real-time PCR shows a significant upregulation of the expression level of the OsCBL1 gene in the susceptible rice variety Nipponbare 24 h after inoculation (Figure 1). This further demonstrates that OsCBL1 is involved as a negatively regulating factor in the rice's defense response against bacterial leaf rot. Example 2: Functional analysis of the OsCBL1 gene in the context of the rice resistance response against bacterial leaf rot. 2.1 Cultivation of the rice material. The seeds of the wild type Nipponbare, the rice plants with gene knockout of OsCBL1 (KO1, KO2) as well as rice plants with overexpression of OsCBL1 (OE-1, OE2) are each sown in nutrient medium and grown under the same cultivation conditions as in Example 1 in the climate chamber. Each line is grown in 20 pots with three plants each. 2.2. Construction of the CRISPR / Cas9 Knockout Vector. The construction of the knockout vector targeting the OsCBL1 gene is based on the vector pYLCRISPR / Cas9Pubi-H (MaX, ZhangQ, ZhuQu. a. Robust CRISPR / Cas9 system for convenient, 25 high-efficiency multiplex genome editing in monocot and dicot plants. Molecular Plant, 2015, 8(8):1274-1284). The nucleotide sequence of the OsCBL1 gene corresponds to SEQIDNO:1, with the coding region occupying nucleotide positions 1 to 642. The selection of the target site is based on a sequence in the first exon of the OsCBL1 gene, resulting in the following target sequence of the sgRNA: BE2026 / 7372 10 5'-CCGGGGTACGAGGACCCCGT-3'(SEQIDNO:3).This target sequence extends over nucleotide positions 31 to 53 of SEQIDNO:1 immediately downstream of the start codon ATG and corresponds to the coding region of amino acid residues 11 to 17 at the N-terminus of the OsCBL1 protein with the sequence PGYEDPV.The selection of these 5 target sites is justified by the fact that insertion or deletion mutations at the N-terminal region of the coding region lead to a frameshift, resulting in premature termination of translation and complete destruction of the biological function of the OsCBL1 protein. The expression cassette of the sgRNA with the 10 aforementioned target sequences is ligated with the vector pYLCRISPR / Cas9Pubi-H cleaved by the restriction enzyme BsaI, yielding the recombinant expression vector pYLCRISPR / Cas9Pubi-H-OsCBL1. 2.3 Molecular Identification of Transgenic Plants 15 Genomic DNA is extracted from young rice leaves using the CTAB method. To identify positive transgenic plants, PCR analysis is performed using specific primers for the hygromycin resistance gene (Hyg-F / R) and specific primers for the gene knockout (cblcr-f / r). Sequence analysis of the PCR products is performed on both materials with gene knockout, followed by sequence comparison to a reference sequence.In both plants with overexpression, the expression level of the OsCBL1 gene is determined by quantitative real-time PCR under the conditions of embodiment 1, whereupon a selection of lines 25 with significantly increased gene expression is made for subsequent experiments. SEQIDNO:8:Hyg-F(5'-CAAAGATCGTTATGTTTATCGGCACT-3') SEQIDNO:9:Hyg-R(5'-TTGGCGACCTCGTATTGGGAA-3') SEQIDNO:10:cblcr-f(5'-TGTAAGGGTAGGTGGAGGG-3') SEQIDNO:11:cblcr-r(5'-GAATGCGGCAAGGTCTAA-3')30 2.4 Inoculation with the pathogen of bacterial leaf rot and BE2026 / 7372 11 Resistance testing The inoculation by leaf cut method takes place at the tillering stage of the rice. The bacterial strain used is adjusted to an OD600 value of 0.5 before approximately 2 to 3 cm of the 5 is cut with sterile scissors moistened with the bacterial suspension. The leaf tip is cut off. Ten plants with three leaves each are inoculated per line, with one inoculation using sterile water serving as a negative control.The clinical picture and the measurement of lesion lengths are performed 14 days after inoculation. At least thirty sheets are measured per line to calculate a mean value. 2.5 Results and Evaluation The results of PCR identification show that both the OsCBL1 gene knockout plants (KO1, KO2) and the overexpression plants (OE-1, OE2) amplify specific bands for the OsCBL1 gene knockout and for the hygromycin resistance gene. In PCR testing with the OsCBL1 knockout-specific primers, target bands are amplified in both the wild type and the knockout lines. The purified PCR products are sequenced and analyzed; 20 insertion mutations (+1 bp) or deletion mutations (−1 bp) are detected at the target site (Figure 2A). In testing for the hygromycin resistance gene, no amplification bands are detectable in the wild type control, whereas positive Overexpression plants exhibit the corresponding target bands.25 The results of quantitative real-time PCR show that the expression level of the OsCBL1 gene in OE-1 is 15 times and in OE2 2.6 times the wild-type level (Figure 2B), confirming successful insertion and high expression of the overexpression vector. 30 As shown in Figure 3, the average lesion length in wild-type Nipponbare 14 days after inoculation is 11.00 cm. The mean lesion lengths of the knockout lines KO1 and KO-2 are 8.00 cm and 6.70 cm respectively, with a very significant difference from the wild type (P < 0.01). The overexpression lines OE-1 and OE2 show average lesion lengths of 12.50 cm and 12.20 cm respectively. on, whereby a significant elongation of the lesions is observed in comparison to the wild type (P<0.001 or P<0.0001). Example 3: Determination of reactive oxygen species in transgenic OsCBL1 rice before and after inoculation with the pathogen of bacterial leaf rot 10 3.1. Cultivation and Inoculation of the Rice Material. The cultivation conditions of the rice are carried out according to the specifications of the exemplary embodiment. 1. At the tillering stage, inoculation with the strain PXO99 is carried out using the leaf cutting method at a bacterial concentration of OD600 = 0.515 by cutting off approximately 2 to 3 cm of the leaf tip with sterile scissors moistened with the bacterial suspension; treatment with sterile water serves as a negative control. Leaf samples are taken from an area of ​​2 cm below the inoculation site at 0 h, 24 and 48 h after inoculation. Three leaves are taken per time point and line, with three biological replicates being established. 3.2. Histochemical staining agent NBT. The NBT staining is carried out according to procedures known from the literature with the following steps: 25 (1) Sample collection: Rice leaves are taken from the 2 cm area below the inoculation site at different times before and after inoculation, with three leaves per line and time point. The removed leaves are soaked overnight in sterile water to eliminate mechanically caused measurement errors. BE2026 / 7372 13 (2) Preparation of the staining solution: An NBT staining solution with a concentration of 0.5 mg / mL is freshly prepared using 10 mmol / LPBS buffer (pH 7.8) and stored protected from light. (3) Staining procedure: The leaf sections, which have been soaked overnight, are transferred to reaction vessels containing NBT staining solution and completely covered by the solution. To infiltrate the staining substance into the leaf tissue, a vacuum infiltration is carried out by evacuation for 15 minutes using a vacuum pump, before light-protected incubation at room temperature for 1 minute.(4) Decolorization: After draining the NBT solution, a 95% ethanol solution with an addition of 5% glacial acetic acid is added, and the sample is decolorized in a boiling water bath for 15 minutes. The decolorizing solution is exchanged once or twice during the process until the green leaf pigments are completely removed. 3.3 Evaluation and Documentation: The decolorized leaves are placed on agar plates, which are placed on white filter paper. The distribution and intensity of the blue precipitates are photographically documented using a camera. The staining results are classified according to the extent and depth of the blue precipitates into the following levels: no staining (−), 20 no blue precipitates on the leaf, agreement with the non-inoculated control; weak staining (+), isolated small blue spots around the inoculation site; medium staining (++), pronounced blue precipitates in the vicinity of the inoculation site; strong staining (+++), large dark blue precipitates around the inoculation site 25 with spreads along the leaf nerves. 3.4. Results and Evaluation: The results of the NBT histochemical staining.

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