A gene and protein combination containing RCD1 and RCD2 and its application

Abstract

The application belongs to the technical field of agricultural genetic engineering, and particularly relates to a gene combination containing RCD1 and RCD2, a protein combination and application thereof. The application provides a codon-optimized gene combination, which comprises an RCD1 gene and an RCD2 gene. The gene combination is beneficial to expression interaction in plants, and further improves the disease resistance of the plants. Furthermore, the application provides a protein combination encoded by the gene combination, a biological material containing the gene combination and application of the biological material in improving the disease resistance of plants and / or cultivating disease-resistant plants. Experiments prove that by introducing the exogenous RCD1 gene and the RCD2 gene into tobacco and rice respectively, the disease resistance of the tobacco to pepper phytophthora disease is improved, and the disease resistance of the rice to bacterial leaf blight is improved without affecting the agronomic traits of the rice.

Description

Technical Field

[0001] This invention belongs to the field of agricultural genetic engineering technology, specifically relating to a gene combination and protein combination containing RCD1 and RCD2 and their applications. Background Technology

[0002] RCD1 and RCD2 are allotype recognition genes in the fungus *Neurospora crassa*. When a *Neurospora crassa* race carrying RCD1 and a *Neurospora crassa* race carrying RCD2 undergo cell fusion, the RCD1 and RCD2 proteins recognize and activate each other, polymerize, and perforate the cell membrane, leading to cell death. The mechanism of allotype recognition leading to cell death in *Neurospora crassa*, represented by RCD1 and RCD2, is widespread in fungi and helps prevent the spread of pathogens such as fungal viruses among fungi.

[0003] Currently, people have increasingly higher requirements for the yield and variety of crops such as grains. Therefore, when breeding disease-resistant plants such as rice, yield needs to be considered simultaneously. In existing technologies, researchers mostly discover and clone new disease-resistant genes from plants to promote research on plant disease resistance and to breed disease-resistant varieties. However, the above methods require a huge amount of human and material resources and are costly. If RCD1 and RCD2 could directly polymerize and interact in plants to enhance their disease resistance, it would greatly save human resources and costs. However, there are no research reports on the effective interaction of RCD1 and RCD2 genes in plants. Summary of the Invention

[0004] The purpose of this invention is to provide a gene combination and protein combination containing RCD1 and RCD2 and their application. By optimizing the codons of the allosome recognition genes RCD1 and RCD2 of Neurospora crassa, it is beneficial for them to interact and take effect in plants, thereby improving the disease resistance of plants without affecting the agronomic traits of the plants themselves.

[0005] This invention provides a gene combination comprising the RCD1 gene and the RCD2 gene;

[0006] The nucleotide sequence of the RCD1 gene is shown in SEQ ID NO.1;

[0007] The nucleotide sequence of the RCD2 gene is shown in SEQ ID NO.2.

[0008] The present invention also provides a protein combination encoded by the gene combination described above, the protein combination including RCD1 protein and RCD2 protein;

[0009] The amino acid sequence of the RCD1 protein is shown in SEQ ID NO.3;

[0010] The amino acid sequence of the RCD2 protein is shown in SEQ ID NO.4.

[0011] The present invention also provides a biomaterial comprising a recombinant vector containing the gene combination described in the above technical solution or an engineered bacterium containing the recombinant vector.

[0012] Preferably, the recombinant vector further includes a promoter;

[0013] The promoter is Xa23-pro.

[0014] The present invention also provides the application of the gene combination, protein combination, or biological material described in the above-mentioned technical solutions in improving plant disease resistance and / or cultivating disease-resistant plants.

[0015] Preferably, the plant includes tobacco and / or rice.

[0016] Preferably, the disease resistance includes resistance to diseases caused by Phytophthora capsici and / or resistance to diseases caused by Blight of the white leaf blight fungus.

[0017] This invention also provides a method for improving plant disease resistance and / or cultivating disease-resistant plants, comprising:

[0018] The gene combination described in the above technical solution is introduced into the target plant to enhance the plant's disease resistance and / or cultivate disease-resistant plants.

[0019] Preferably, the introduction includes injecting a transformation solution containing an RCD1 gene and an RCD2 gene expression vector into the target plant.

[0020] Preferably, the target plant includes tobacco and / or rice.

[0021] Beneficial effects:

[0022] This invention provides a gene combination comprising an RCD1 gene and an RCD2 gene; the nucleotide sequence of the RCD1 gene is shown in SEQ ID NO.1; and the nucleotide sequence of the RCD2 gene is shown in SEQ ID NO.2. Targeting plant expression characteristics, this invention provides codon-optimized RCD1 and RCD2 genes. By regulating the expression of the above gene combination in plants, the disease resistance of plants is improved without affecting their agronomic traits.

[0023] Based on the aforementioned technical advantages, this invention further provides gene-encoded protein combinations, biomaterials containing gene combinations, and their applications in enhancing plant disease resistance and / or cultivating disease-resistant plants. Experiments have shown that introducing exogenous RCD1 and RCD2 genes into tobacco improves tobacco's resistance to Phytophthora capsulatum; introducing exogenous RCD1 and RCD2 genes into rice improves rice's resistance to bacterial blight without affecting its agronomic traits. Attached Figure Description

[0024] 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.

[0025] Figure 1 The different treatments in Example 2 induced the HR response in tobacco leaves;

[0026] Figure 2 This illustrates the effects of different treatments on resistance to Phytophthora capsici in Example 2.

[0027] Figure 3 The different treatments in Example 2 induced the upregulation of tobacco disease resistance-related genes;

[0028] Figure 4 The expression levels of genes induced by the PXO99 strain of *Bacillus subtilis* under different treatments in Example 3;

[0029] Figure 5 The resistance of rice to bacterial blight strain PXO99 in different treatments in Example 3;

[0030] Figure 6 The phenotypes of rice under different treatments in Example 3 are shown. Detailed Implementation

[0031] The present invention provides a gene combination comprising an RCD1 gene and an RCD2 gene; the RCD1 gene is a codon-optimized gene, and its nucleotide sequence is shown in SEQ ID NO.1 as 5'-ATGGACAAGTGTTGGT-3'.

[0032] The RCD2 gene described in this invention is a codon-optimized gene, and its nucleotide sequence is shown in SEQ ID NO.2: 5'-ATGGATAACGAAGAGTGGTTTCCGCTGAAACAGACACACTATCCACCGCCAACTATTCCGA-3'. Because codon optimization was performed on the expression characteristics of plants, both the RCD1 and RCD2 genes can be expressed in plants, thereby enhancing their disease resistance. In a specific embodiment of this invention, the online tool for codon optimization is https: / / www.novopro.cn / tools / codon-optimization.html.

[0033] This invention provides a protein combination encoded by the gene combination described in the above-mentioned technical solution, the protein combination comprising RCD1 protein encoded by the RCD1 gene and RCD2 protein encoded by the RCD2 gene; the amino acid sequence of the RCD1 protein is shown in SEQ ID NO.3, and is: MDKCWFTLDNAHYPPPSLDSMRSGHPISPASLGHLIPSLAHLDQIINAKAIEPFPATMDIHGPTIIEDFKWDHSHEYSLSLGGKVPIPLAPAGVPFVDLNVGLGGAFSRSVANYWEFDRLERYIMQPTRSYVQKCIERDEVKRWIAKNKSMMMMGRWEVYMITGIIVARGGGRKKKEKTTGKEFSVEVTVEVPLIVEAGPGGKRNTARQKTWGTSQTGDFVWAVRLAKITKSGLHSDWKMETVFGKTSSFRGQKAIF*; the amino acid sequence of the RCD2 protein is shown in SEQ ID NO.4, and is: MDNEEWFPLKQTHYPPPTI PSMKTGHPTGPISIGHIIPDLRHLDNVINCKGFEPFPPNMDVFTAHYEQCHFGDHLNSEFVVQAKAAAPIKNIVPGVDVTGSAGLHHTNITSDRWEYDSVVEYAVYPTRQYIDRLLESKEVKQYIQKSKKLLGGWCVYMVTGIMVARGGGRNVVSEEKGAGVFGNVGFQVPGIGEFAPEVGWDTKTKTKVNAHHTTDFVCAIRLVKIAKSGLRSSWTMKKVTREF*. These protein combinations can also interact in plants, forming protein multimers that perforate the cell membrane, leading to cell death and thus conferring disease resistance.

[0034] This invention provides a biological material comprising a recombinant vector containing the gene combination described in the above technical solution or an engineered bacterium containing the recombinant vector; the base vector used to construct the recombinant vector is preferably pCAMBIA1300 or PRHE (disclosed in the following literature, He F, Zhang F, Sun W, Ning Y, Wang GL. A Valsatile Vector Toolkit for Functional Analysis of Rice Genes. Rice(NY). 2018 Apr 20;11(1):27. doi:10.1186 / s12284-018-0220-7. Erratumin: Rice(NY). 2018 Sep 7;11(1):49.doi:10.1186 / s12284-018-0238-x.PMID:29679176;PMCID:PMC5910328.);The recombinant vector preferably includes a promoter; the promoter is preferably Xa23-pro; the Xa23-pro is contained in the sequence with accession number KP123634.1 on NCBI, and the nucleotide sequence of the Xa23-pro is preferably as shown in SEQ ID NO.29, specifically: 5'-ctgaggtagctgccacgtcagctaggggatcggcccggccgccacggtggcacaatgtcagcgccagtcccgttggcgctgacacggccaacgt cagcgccaatgtgtttggcgctgaggcgacggcctatttttggttgaagttttggcaggggttagtttcgaaataagtttttccaaaagggtcaatttgtcaaaaaacggctcgtccctgagtcaaagtcttccctattaaattatgcggcatcactaacatcagctactata aaagtcccttccgcgtcactaacatcagctactataaaagtcccttccgaaacatcttcctcccgcatcactaacatcagcttctataaaagcccttccttgttgcatcatctcaaggagctgcaagcacttcctctctggcagcacttcctcatctcaaggagttgcaa-3'.Because Xa23-pro is an inducible promoter, its regulatory sequence expression level is low during normal plant growth. However, when it is affected by a promoter containing the AvrXa23 effector (disclosed in the following literature: Xu Z, Xu X, Wang Y, Liu L, Li Y, Yang Y, Liu L, Zou L, Chen GA varied AvrXa23-like TALE enables the bacterialblight pathogen to avoid being trapped by Xa23 resistance gene in rice. J AdvRes. 2022 Dec; 42:263-272. doi:10.1016 / j.jare.2022.01.007.Epub 2022 Jan) When infected by strains (PMID:36513417; PMCID:PMC9788936.), the regulatory sequence is significantly upregulated. Therefore, Xa23-pro is suitable as a promoter to regulate the co-expression of RCD1 and RCD2 genes, which is beneficial to confer resistance to bacterial blight on plants without affecting normal plant growth.

[0035] This invention provides the application of the gene combination, protein combination, or biological material described in the above-mentioned technical solution in improving plant disease resistance and / or cultivating disease-resistant plants; the disease resistance preferably includes: resistance to diseases caused by Phytophthora capsici and / or resistance to diseases caused by Blight of the plant; more preferably, resistance to tobacco black shank and resistance to rice bacterial blight; the plant preferably includes tobacco and / or rice, more preferably tobacco and rice. This invention does not specifically limit the varieties of tobacco and rice; conventional tobacco or rice varieties in the art can be used. For example, in a specific embodiment of this invention, the tobacco is *Nicotiana benthamiana*, and the rice is *Kitaake*.

[0036] This invention provides a method for enhancing plant disease resistance and / or cultivating disease-resistant plants, comprising: introducing the gene combination described in the above-mentioned technical solution into a target plant to enhance plant disease resistance and / or cultivate disease-resistant plants. The target plant of this invention preferably includes tobacco and / or rice, more preferably tobacco or rice; the introduction preferably includes: injecting a transformation solution containing an RCD1 gene and an RCD2 gene expression vector into the target plant; the components and preparation method of the transformation solution do not have special requirements and can be any method well known in the art.

[0037] Experiments have shown that by using the technical solution provided by this invention, the introduction of exogenous RCD1 and RCD2 genes into tobacco is beneficial to inducing tobacco hypersensitivity (HR) response and reactive oxygen species (ROS) burst, thereby endowing tobacco with resistance to Phytophthora capsulatum. The introduction of exogenous RCD1 and RCD2 genes into rice improves the rice's resistance to bacterial blight without affecting the rice's agronomic traits.

[0038] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a gene combination and protein combination containing RCD1 and RCD2, and their applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1: Sequence optimization of allogeneic recognition genes RCD1 and RCD2 in Neurospora crassa

[0040] The codons of the allosome recognition genes RCD1 and RCD2 of Neurospora crassa were optimized using an online codon optimization tool (https: / / www.novopro.cn / tools / codon-optimization.html) to improve the translation efficiency of RCD1 and RCD2 in plants and obtain higher levels of protein expression. The optimization results are as follows:

[0041] The optimized RCD1 gene sequence for plants is shown in SEQ ID NO.1, and the RCD1 protein sequence encoded by the optimized RCD1 gene is shown in SEQ ID NO.3; the optimized RCD2 gene sequence for plants is shown in SEQ ID NO.2, and the RCD2 protein sequence encoded by the optimized RCD2 gene is shown in SEQ ID NO.4. For ease of explanation, the genes RCD1 and RCD2 mentioned below refer to the optimized RCD1 and RCD2.

[0042] Example 2: Co-expression experiment of Neurospora crassa RCD1 and RCD2

[0043] S1. Carrier Construction:

[0044] The full-length fragments of RCD1 (SEQ ID NO.1) and RCD2 (SEQ ID NO.2) were amplified separately and constructed into the pCAMBIA1300 vector via homologous recombination. The primers for amplifying RCD1 are as follows: P1300-RCD1-F: 5'-CCAAATCGACTCTAGAAAGCTTATGGACAAGTGTTGGTTCACTCT-3' (SEQ ID NO.5); P1300-RCD1-R: 5'-GGGAAATTCGAGCTCGGTACTCAGAAGATGGCCTTCTGACCTCTG-3' (SEQ ID NO.6).

[0045] The primers for amplifying RCD2 are as follows: P1300-RCD2-F: 5'-CCAAATCGACTCTAGAAAGCTTATGGATAACGAAGAGTGGTTTCCG-3' (SEQ ID NO.7); P1300-RCD2-R: 5'-GGAAATTCGAGCTCGGTACTCAGAATTCCCTAGTGACCTTCTTCATTGTC-3' (SEQ ID NO.8);

[0046] The amplification reaction conditions were as follows: 95℃ for 5 min; 95℃ for 30 sec, 58℃ for 30 sec, 72℃ for 30 sec, 34 cycles; 72℃ for 5 min.

[0047] The homologous recombination ligation process was as follows: the pCAMBIA1300 vector was double-digested with Hind III and Kpn I to obtain the digested products; the digested products were ligated with the amplified RCD1 fragment and the amplified RCD2 fragment, respectively, to construct the tobacco expression vectors pCAMBIA1300-RCD1 and pCAMBIA1300-RCD2; pCAMBIA1300-RCD1 and pCAMBIA1300-RCD2 were transformed into Escherichia coli DH5α, respectively, to obtain protein expression vectors containing RCD1 and RCD2.

[0048] S2. Immunological Characterization:

[0049] (1) Co-expression of RCD1 and RCD2 in Nicotiana benthamiana induces hypersensitivity (HR) response in plants:

[0050] The experiment was divided into 5 groups, with each group being a treatment and each treatment being replicated 5 times in parallel. The group expressing RCD1 alone was denoted as RCD1, the group expressing RCD2 alone was denoted as RCD2, the group co-expressing RCD1 and RCD2 was denoted as RCD1+2, the empty vector pCAMBIAsuper1300 (negative control) was denoted as EV, and the positive control that induced the HR response was denoted as Xa23.

[0051] The specific experimental procedure was as follows: The constructed vectors pCAMBAIA1300-RCD1, pCAMBAIA1300-RCD2, and pCAMBAIA1300-Xa23 (positive control) were transformed into Agrobacterium GV3101, respectively. After 16 hours of shaking culture, the bacterial cells were resuspended in infection solution (composed of 10 mM MgCl2, 10 mM morpholinoethanesulfonic acid (MES), and 100 μM acetylsyleugenone) to obtain Agrobacterium containing the RCD1 expression plasmid, Agrobacterium containing the RCD2 expression plasmid, and Agrobacterium containing the Xa23 expression plasmid, respectively. The OD values ​​of each bacterial culture were then recorded. 600 Adjust to 1.0; Agrobacterium containing RCD1 expression plasmid and Agrobacterium containing RCD2 expression plasmid are mixed at a volume ratio of 1:1 to obtain a mixed bacterial solution that can co-express RCD1 and RCD2 in tobacco after co-injection. This mixed bacterial solution is denoted as RCD1+2.

[0052] The primers used to construct pCAMBAIA1300-Xa23 are Xa23-F: 5'-ccaaatcgactctagaaagcttatgttgcatcatctcaagg ag-3' (SEQ ID NO.34); Xa23-R: 5'-gggaaattcgagctcggtacttaaacagggagaataaccatcttgtcgtc-3' (SEQ ID NO.35).

[0053] Agrobacterium containing the RCD1 expression plasmid, Agrobacterium containing the RCD2 expression plasmid, Agrobacterium containing the Xa23 expression plasmid, mixed bacterial culture RCD1+2, empty vector negative control (EV), and positive control Agrobacterium containing the Xa23 expression plasmid were injected into the same four-week-old Tobacco Benedict leaf. Each treatment was based on an injection area of ​​approximately a circular region with a diameter of 2 cm.

[0054] Twenty-four hours after injection, tobacco leaves were observed and photographed under white light and ultraviolet (UV) light, respectively. The results are shown below. Figure 1 The left and middle images ( Figure 1 The left image shows the result of a photograph taken under white light; the middle image shows the result of a photograph taken under violet light; in Figure 1In each figure, the white line in the lower right corner represents a scale bar of 1 cm in length; RCD1 represents the result of RCD1 expressed alone; RCD2 represents the result of RCD2 expressed alone; RCD1+2 represents the result of co-expression of RCD1 and RCD2; EV represents the result of negative control; Xa23 represents the result of positive control.

[0055] Depend on Figure 1 It can be seen that the regions co-expressing RCD1 and RCD2 (i.e., RCD1+2) reacted in the same way as the positive control Xa23 region, both of which induced HR responses in tobacco cells; while the responses of RCD1 or RCD2 expressed alone were consistent with those of the empty vector (EV), and did not induce HR responses in tobacco cells.

[0056] (2) Co-expression of RCD1 and RCD2 induces reactive oxygen species (ROS) burst in Nicotiana benthamiana - DAB staining

[0057] Following the method described in (1) above, Agrobacterium containing the RCD1 expression plasmid, Agrobacterium containing the RCD2 expression plasmid, Agrobacterium containing the Xa23 expression plasmid, Agrobacterium containing the RCD1+2 expression plasmid, the empty vector negative control (EV), and the positive control Agrobacterium containing the Xa23 expression plasmid were injected into the same four-week-old Tobacco Benedict leaf. Each treatment was based on an injection area of ​​approximately a circular region with a diameter of 2 cm.

[0058] Fifteen hours after injection, each injected tobacco leaf was immersed in 20 mL of DAB dye solution and incubated horizontally in the dark for 8 hours. Then, it was placed in 95% ethanol for decolorization and incubated horizontally for 48 hours. The results were then photographed under white light. (See attached image). Figure 1 The right image in the middle ( Figure 1 The right image shows the DAB staining results; the white line in the lower right corner represents a scale bar with a length of 1 cm; RCD1 represents the result of RCD1 expression alone; RCD2 represents the result of RCD2 expression alone; RCD1+2 represents the result of co-expression of RCD1 and RCD2; EV represents the result of the negative control; Xa23 represents the result of the positive control.

[0059] Depend on Figure 1 As shown in the right figure, co-expression of RCD1 and RCD2 (i.e., RCD1+2) and the positive control Xa23 both induced ROS bursts in tobacco cells, while expression of RCD1 or RCD2 alone, consistent with the empty vector (EV), did not exhibit this phenomenon. Therefore, co-expression of RCD1 and RCD2 in tobacco cells can induce ROS bursts, just like the positive control Xa23.

[0060] (3) Co-expression of RCD1 and RCD2 confers resistance to Phytophthora benthamiana.

[0061] According to the experimental method in (1) above, Agrobacterium containing RCD1 expression plasmid, Agrobacterium containing RCD2 expression plasmid, Agrobacterium containing Xa23 expression plasmid, Agrobacterium containing RCD1+2 expression plasmid, empty vector negative control (EV), and positive control Agrobacterium containing Xa23 expression plasmid were injected into four-week-old Tobacco Benedictine leaves. Each treatment was based on an injection area of ​​approximately a circular area with a diameter of 2 cm. Specifically, EV and RCD1 (treatment 2) were injected into two circular areas of equal area on the same leaf, EV and RCD2 (treatment 3) were injected into two circular areas of equal area on the same leaf, EV and RCD1+2 (treatment 1) were injected into two circular areas of equal area on the same leaf, and EV and Xa23 (treatment 4) were injected into two circular areas of equal area on the same leaf. Each treatment was performed in 5 parallel replicates.

[0062] Inoculate with *Phytophthora capsici* LT263 15 hours after injection (to ensure the activity and sufficient pathogenicity of *Phytophthora capsici*, use colonies that cover approximately 80% of the PDA plate area). The specific method is as follows:

[0063] Leaflets of *Tobacco Bengal* treated with *Agrobacterium* were removed and placed in petri dishes containing filter paper moistened with ultrapure water. A 3mm diameter LT263 bacterial block was inoculated at the center of the inoculation site, and the dishes were then sealed with plastic wrap. After inoculation, each sample was placed in a 28℃ incubator for dark incubation. Depending on the size of the tobacco leaves, photographs were taken under UV light at 18–36 hpi (hours post-incubation) and data were collected. Additionally, using the area of ​​a square that encloses the largest lesion as a reference, samples of the same area were taken from each lesion (e.g., assuming the largest lesion is 1cm). 2 Therefore, all samples are taken from a 1cm radius centered on the inoculation point. 2 Leaf samples were collected, and DNA was extracted from the samples. The following primers were used to perform qPCR detection of the EF1α gene in tobacco and the tubulin gene in *Phytophthora capsici*, respectively:

[0064] qRT-NbEF1α-F:5'-AGAGGGCCCTCAGACAAAC-3' (SEQ ID NO. 30);

[0065] qRT-NbEF1α-R:5'-TAGGTCCAAAGGTCACAA-3' (SEQ ID NO. 31);

[0066] qRT-Pctubulin-F: 5'-CAGAGGGTGCTGAGCTTATTGACT-3' (SEQ ID NO. 32);

[0067] qRT-Pctubulin-R: 5'-GAGAGTGGGTGATCTGGAAACCC-3' (SEQ ID NO. 33);

[0068] Of these two genes, EF1α was used as an internal reference gene, and 2... -ΔΔCt The relative expression level of the tubulin gene was calculated using a method to serve as a reference value for *Phytophthora capsici* biomass. Results for lesion area and *Phytophthora capsici* biomass are shown in Tables 1 and 2. Figure 2 (exist Figure 2 In the diagram, RCD1 represents the result of RCD1 expression alone; RCD2 represents the result of RCD2 expression alone; RCD1+2 represents the result of co-expression of RCD1 and RCD2; EV represents the result of the negative control; Xa23 represents the result of the positive control; and the white line in the lower right corner represents a scale bar with a length of 1 cm. Figure 2 The first row of images shows the experimental results of each treatment under ultraviolet light; Figure 2 The left graph in the middle section represents the lesion area under different treatments, and the right graph represents the biomass of Phytophthora capsici under different treatments.

[0069] Table 1. Lesion area under different treatments, unit: cm² 2

[0070]

[0071] Table 2. Phytophthora capsici biomass under different treatments

[0072] - EV RCD1+2 RCD1 RCD2 Repeat 1 4.792096 0.000801681 3.625214 6.054623 Repeat 2 3.421285 0.002480221 6.194406 3.007069 Repeat 3 3.005939 0.313855 2.204427 4.343249

[0073] From Table 1, Table 2 and Figure 2 It can be seen that when *Phytophthora capsici* is co-expressed with RCD1 and RCD2, the lesion area and biomass in the expression region of *Phytophthora capsici* are significantly smaller than those in the expression regions of the positive control Xa23 compared to those in the expression regions of RCD1 alone, RCD2 alone, and the empty vector (EV). Therefore, co-expression of RCD1 and RCD2 can confer resistance to *Phytophthora capsici* in *Nicotiana benthamiana*.

[0074] (4) Co-expression of RCD1 and RCD2 induces upregulation of genes related to resistance to Nicotine Benigne.

[0075] The specific experimental method was as follows: Four treatment groups were set up, namely, Agrobacterium tumefaciens expressing RCD1 (denoted as RCD1), Agrobacterium tumefaciens expressing RCD2 (denoted as RCD2), and Agrobacterium tumefaciens expressing EV plasmid (denoted as EV) were injected into Nicotiana benthamiana individually, and Agrobacterium tumefaciens expressing RCD1 and RCD2 plasmids were injected together (i.e., RCD1 and RCD2 were mixed at a volume ratio of 1:1, denoted as RCD1+2). The RCD1+2 sample was the experimental group, and RCD1, RCD2, and EV were the control group. Each treatment group was performed in triplicate. In the four treatment groups, each leaf was injected according to the treatment group, with one leaf per treatment group. Only the treatment group that injected both RCD1 and RCD2 was injected with one leaf containing both bacterial cultures (RCD1 and RCD2), while the others were injected with one culture per leaf.

[0076] RNA extraction and quantitative reverse transcription PCR analysis: Tobacco leaves containing RCD1, RCD2, RCD1+2, and EV were collected 15 h after Agrobacterium injection and rapidly frozen in liquid nitrogen. After grinding in liquid nitrogen, RNA was extracted. Total RNA was extracted from different samples using the RNAprepPure Plant Kit (TianGen Biotech; China). RNA was purified from 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).

[0077] The primers for amplifying the LOX gene are: qRT-LOX / F:5'-aaaacctatgcctcaagaac-3' (SEQ ID NO. 9) and qRT-LOX / R:5'-actgctgcataggctttgg-3' (SEQ ID NO. 10); the primers for amplifying the PAL gene are: qRT-PAL / F:5'-gttat gctcttagaacgtcgccc-3' (SEQ ID NO. 11) and qRT-PAL / R:5'-ccgtgtaatgccttgtttcttga-3' (SEQ ID NO. 12); and the primers for amplifying the PR1b gene are: qRT-PR1b / F:5'-gtggacactatactcaggtg-3' (SEQ ID NO. 13) and qRT-PR1b / R:5'-tccaacttggaatcaaaggg-3' (SEQ ID NO. 14). NO.14); The primers for amplifying the PR2b gene are: qRT-PR2b / F:5'-aggtgtttgct atggaatgc-3' (SEQ ID NO.15) and qRT-PR2b / R:5'-tctgtacccaccatcttgc-3' (SEQ ID NO.16).

[0078] Use 2 -ΔΔCT The expression levels of PR1b, PR2b, PAL, and LOX genes in different treatments were calculated, and the results are shown in Tables 3-6. Figure 3 ,exist Figure 3 In the table, RCD1 represents the result of RCD1 expression alone; RCD2 represents the result of RCD2 expression alone; RCD1+2 represents the result of co-expression of RCD1 and RCD2; EV represents the result of negative control; NbPR1b represents the expression level of PR1b; NbPR2b represents the expression level of PR2b; NbPAL represents the expression level of PAL; NbLOX represents the expression level of LOX; significance “*” indicates P<0.05; “**” indicates P<0.01; “***” indicates P<0.001; “****” indicates P<0.0001.

[0079] Table 3. LOX gene expression levels in different treatments

[0080] - EV RCD1 RCD2 RCD1+2 Repeat 1 0.03433904 0.5097291 2.7022 14.27088 Repeat 2 0.08520445 0.7843708 5.191552 14.76476 Repeat 3 0.3585153 0.9285614 3.281273 12.57612

[0081] Table 4. Expression levels of the PAL gene in different treatments

[0082] - EV RCD1 RCD2 RCD1+2 Repeat 1 0.8133568 0.5872876 1.946517 2.51692 Repeat 2 0.7896305 0.280207 1.96516 2.489612 Repeat 3 0.6698019 0.7156582 2.39864 2.363766

[0083] Table 5. Expression levels of the PR1b gene in different treatments.

[0084] - EV RCD1 RCD2 RCD1+2 Repeat 1 1.055346 0.2845837 3.015423 8.91034 Repeat 2 0.3940709 0.1881303 2.82815 8.44573 Repeat 3 0.5672318 1.642141 3.307323 6.73664

[0085] Table 6. Expression levels of the PR2b gene in different treatments

[0086] - EV RCD1 RCD2 RCD1+2 Repeat 1 2.28735 1.809993 5.201949 13.96182 Repeat 2 3.348259 1.47223 5.864811 12.15682 Repeat 3 1.845477 4.212994 6.191238 13.59672

[0087] From Tables 3-6 and Figure 3 The results showed that in tobacco samples injected with RCD1+2, the expression levels of PR1b, PR2b, PAL, and LOX disease resistance genes were significantly increased, significantly higher than those in the control groups injected with RCD1 and EV alone. While the control group injected with RCD2 alone also showed some degree of upregulation, the level and number of upregulated genes were lower than in the experimental group. Therefore, the co-expression of RCD1 and RCD2 activates the expression of plant disease resistance genes, demonstrating that the co-expression of RCD1 and RCD2 genes exhibits plant R-gene immune characteristics.

[0088] In summary, the RCD1 and RCD2 proteins of Neurospora crassa possess the basic characteristics of plant disease resistance proteins.

[0089] Example 3 uses the inducible promoter (Xa23-pro) of rice gene Xa23 to regulate the co-expression of Neurospora crassa RCD1 and RCD2, thereby conferring bacterial blight resistance to rice without affecting its agronomic traits.

[0090] Selection of S1, RCD1, and RCD2 promoters and construction of co-expression vectors

[0091] As shown in Example 2, co-expression of RCD1 and RCD2 can induce a disease resistance (HR) response in plants, leading to necrosis of the expression region and thus conferring disease resistance. However, food crops such as rice need to maintain a balance between disease resistance and agronomic traits, and cannot sacrifice agronomic characteristics while pursuing disease resistance. Therefore, the promoters regulating the co-expression of RCD1 and RCD2 need to be carefully selected.

[0092] The inducible promoter Xa23-pro (GenBank accession number KP123634.1) of the rice gene Xa23 exhibits low expression levels of its regulatory sequence during normal growth. However, upon infection by a blight strain containing the AvrXa23 effector, its regulatory sequence is significantly upregulated. This pathogen-induced expression characteristic makes it suitable as a promoter for regulating the co-expression of RCD1 and RCD2. Xa23-pro and RCD1-RCD2 (i.e., RCD1 and RCD2 co-expression) were constructed into the stable rice expression vector PRH E using the T4 ligation method. The vector construction steps are as follows:

[0093] 1) First, four fragments were amplified: RCD1-CDS, RCD2-CDS, Nos-Terminator, and Xa23-promoter. The primer sequences used were as follows: Xa23-pro F: 5'-CCCAAGCTTGGGctgaggtagctgccacgtca-3' (SEQ ID NO.35); Xa23-pro R: 5'-Ttgcaactccttgagatgaggaagtg-3' (SEQ ID NO.36); RCD1-CDS-F: 5'-cacttcctcatctcaag gagttgcaaATGGACAAGTGTTGGTTCACTCT-3' (SEQ ID NO.37); RCD1-CDS-R: 5'-CTTTATTGC CAAATGTTTGAACGATCTCAGAAGATGGCCTTCTGACCTCTG-3' (SEQ ID NO.37). ID NO.38); RCD2-CDS-F:5'-cacttcctcatctcaaggagttgcaaATGGATAACGAAGAGTGGTTTCCG-3'(SEQ ID NO.39); RC D2-CDS-R:5'-CTTTATTGCCAAATGTTTGAACGATCTCAGAATTCCCTAGTGACCTTCTTCATTG-3'(SEQ ID NO.40); Nos-TF: 5'-GATCGTTCAAACATTTGGCAATAAAG-3' (SEQ ID NO. 41); Nos-TR-Sac I: 5'-CGAGCTCGGATCTAGTAACATAGATGACACCGC-3' (SEQ ID NO. 42); Nos-TR-BamHI: 5'-CGCGGATCCGCGGATCTAGTAACATAGATGACACCGC-3' (SEQ ID NO.43).

[0094] 2) Subsequently, PCR was used to fuse the two fragments. The first fragment was “Xa23-promoter+RCD1-CDS+Nos-Terminator” and the second fragment was “Xa23-promoter+RCD2-CDS+Nos-Terminator”. After obtaining the two fusion fragments, the PRHE vector and the fused Xa23pro-RCD1-NosT fragment were first digested with Hind III and Sac I. The fragment was then ligated into the PRHE vector using the T4 ligation method. After sequencing confirmed that the recombinant plasmid was correct, it was amplified by E. coli and a large amount of plasmid was extracted. Then, the recombinant PRHE vector and the fused second fragment Xa23pro-RCD2-NosT were digested with Sac I and Ba mHI. The fragment was then ligated into the recombinant PRHE vector using the T4 ligation method.

[0095] 3) After two T4 ligations, the PRHE-Xa23pro-RCD1+2 vector was finally obtained and used for stable expression in rice. The vector was transformed into rice by Agrobacterium-mediated transformation.

[0096] S2. Identification of rice disease resistance phenotypes

[0097] (1) qRT-PCR detection showed that RCD1-RCD2 (i.e., co-expression of RCD1 and RCD2) was significantly induced by Bacillus subtilis strain.

[0098] The *Bacillus thuringiensis* strain AH28 does not contain the effector AvrXa23, while PXO99 does. Therefore, strain AH28 was used as a control. The specific experimental method was as follows: *Bacillus thuringiensis* strains PXO99 and AH28 were cultured separately in NB medium by shaking. When the OD of the bacterial culture reached... 600 When the OD value is 0.5, the bacterial culture is centrifuged at 4000 rpm for 10 min, and then the bacterial cells are collected. The bacterial cells are resuspended in ddH2O to wash the culture medium, and then centrifuged again at 4000 rpm for 10 min, and the bacterial cells are collected again. Then, the collected bacterial cells are adjusted to OD value using ddH2O again. 600 =0.5, reserved;

[0099] Using a 1 mL syringe, inject 0.2 mL of Bacillus subtilis PXO99 (denoted as RCD1+299), 0.2 mL of AH28 (denoted as RCD1+228), and 0.2 mL of ddH2O (denoted as RCD1+2H) onto the upper surface of the flag leaf of the RCD1+2 strain, respectively. Separately, inject 0.2 mL of Bacillus subtilis PXO99 (denoted as Kitaake 99) onto the upper surface of the flag leaf of the Kitaake strain. Three technical replicates were injected for each leaf, and three biological replicates were injected for each rice plant.

[0100] Samples were taken 2 days after injection, and RNA extraction, reverse transcription, and quantitative analysis were performed using the same methods as in Example 2. The primers for amplifying the RCD1 gene were: qRT-RCD1-F:5'-GGCAAGAGGTGGAGGCAGAAAGAA-3' (SEQ ID NO. 17) and qRT-RCD1-R:5'-CGCCTGGACCAGCTTCGACTATGA-3' (SEQ ID NO. 18); the primers for amplifying the RCD2 gene were: qRT-RCD2-F:5'-GTATCATGGTTGCTAGAGGTGGCG-3' (SEQ ID NO. 19) and qRT-RCD2-R:5'-CCTTGGTCTTAGTCTTGGTGTCCC-3' (SEQ ID NO. 20); and the primer for amplifying the internal reference gene OsActin was: qRT-OsActin-F:5'-CAGGCCGTCCTCTCTCTGTA-3' (SEQ ID NO. 19). NO.21) and qRT-OsActin-R:5'-AAGGATAGCATGGGGGAGAG-3' (SEQ ID NO.22), the expression results of RCD1 and RCD2 genes in different treatments are shown in Tables 7 and 8. Figure 4 The first row of images and Figure 6 ,exist Figure 4 In the diagram, RCD1, RCD2, and RCD1+2 represent rice lines stably expressing RCD1, RCD2, and co-expressing RCD1 and RCD2, respectively. Kitaake represents the wild-type material of these lines, i.e., the genetic background. 99 indicates inoculation with *Bacillus subtilis* strain PXO99, and 28 indicates inoculation with *Bacillus subtilis* strain AH28. The left figure in the first row shows the expression level of the RCD1 gene in different treatments; the right figure in the first row shows the expression level of the RCD2 gene in different treatments; in... Figure 4 In the diagram, RCD1+228 represents the result after injection of AH28; RCD1+299 represents the result after injection of PXO99. Figure 6 WT in the text indicates Kitaake wild-type material.

[0101] Table 7. RCD1 gene expression levels in different treatments

[0102] - RCD1+228 RCD1+299 RCD1+2H Kitaake99 Repeat 1 1.353903 49.18957 1.053125 0.3948591 Repeat 2 2.676404 45.8978 0.4761641 0.2156544 Repeat 3 1.044121 40.0108 1.640354 0.2759586

[0103] Table 8. RCD1 gene expression levels under different treatments

[0104] - RCD1+228 RCD1+299 RCD1+2H Kitaake99 Repeat 1 1.113256 37.5984 1.256651 0.225616 Repeat 2 1.6282 39.26599 0.846165 0.2262 Repeat 3 1.49861 34.21982 0.961115 0.1959819

[0105] From Tables 7 and 8 Figure 4 and Figure 6 It can be seen that the transformed line RCD1+2 showed low basal expression during normal growth (without inoculation with Bacillus subtilis strain PXO 99). However, after injection with Bacillus subtilis strain PXO 99, the expression of RCD1 and RCD2 was significantly upregulated, while no upregulation was observed in the control groups injected with Bacillus subtilis strain AH28 and water. This indicates that Xa23-pro can be induced by Bacillus subtilis strain PXO 99 to regulate the co-expression of RCD1 and RCD2, achieving low basal expression during normal growth, thus not significantly affecting the agronomic traits of rice. Only when infected with Bacillus subtilis strain PXO 99 can the expression of RCD1 and RCD2 be significantly upregulated, thereby conferring disease resistance to rice.

[0106] (2) Co-expression of RCD1 and RCD2 induces upregulation of rice disease resistance-related genes.

[0107] Experimental method: The same method as in Example 3S2(1) was used to prepare the infecting bacterial solution and inject it into rice. Samples were taken 2 days after sampling, and RNA extraction, reverse transcription, and quantitative analysis were performed using the same methods as in Example 2. The primer sequences for amplifying the OsPAL gene were qRT-OsPAL / F:5'-ctacccgctgatgaagaagc-3' (SEQ ID NO. 23) and qRT-OsPAL / R:5'-gaaccttgttcagctcctcg-3' (SEQ ID NO. 24); the primer sequences for amplifying the OsPBZ gene were qRT-OsPBZ / F:5'-atgaagctcaaccctgctgt-3' (SEQ ID NO. 25) and qRT-OsPBZ / R:5'-tgagcttgcccaccttactt-3' (SEQ ID NO. 26); and the primer sequence for amplifying the OsKS4 gene was qRT-OsKS4 / F:5'-caaattagggatgggaggctag-3' (SEQ ID NO. 24). NO.27) and qRT-OsKS4 / R:5'-acattcagggaagcatggag-3' (SEQ ID NO.28), the expression levels of each gene in different treatments are shown in Tables 9-11 and Figure 4 The second row of images (in) Figure 4 In the middle, the second row, left panel shows the expression level of the OsPBZ gene under different treatments; the second row, middle panel shows the expression level of the OsKS4 gene under different treatments; the second row, right panel shows the expression level of the OsPAL gene under different treatments; Figure 4 In the diagram, RCD1+228 represents the result after injection of AH28; RCD1+299 represents the result after injection of PXO99.

[0108] Table 9. Expression levels of the OsKS4 gene in different treatments.

[0109] - RCD1+228 RCD1+299 RCD1+2H Kitaake99 Repeat 1 0.6099 3.07648 0.68143 0.41021 Repeat 2 1.27409 2.44452 0.7457 0.4414 Repeat 3 0.67203 4.36448 0.76365 0.27898

[0110] Table 10. Expression levels of the OsPBZ gene in different treatments.

[0111] - RCD1+228 RCD1+299 RCD1+2H Kitaake99 Repeat 1 1.386168 3.02787 0.5605347 0.5289574 Repeat 2 1.788647 10.0385 0.4311674 0.5581664 Repeat 3 1.539991 6.603504 0.4711627 0.2478917

[0112] Table 11. Expression levels of the OsPAL gene in different treatments

[0113] - RCD1+228 RCD1+299 RCD1+2H Kitaake99 Repeat 1 1.397107 3.392005 0.001571127 0.3033761 Repeat 2 2.888362 3.065201 0.02656113 0.1777523 Repeat 3 1.000039 7.226714 0.03980539 0.1895732

[0114] From Tables 9-11 and Figure 4 It can be seen that the transformed line RCD1+2 showed low expression of disease resistance-related genes during normal growth (without inoculation with Bacillus subtilis strain PXO99). However, after injection with Bacillus subtilis strain PXO99, the expression of RCD1 and RCD2 was significantly upregulated. Furthermore, the upregulation of RCD1 and RCD2 was accompanied by a significant upregulation of disease resistance-related genes OsPBZ, OsKS4, and OsPAL. In contrast, the control group RCD1, RCD2, and WT showed no significant upregulation of disease resistance-related genes. Therefore, Xa23-pro can induce the co-expression of RCD1 and RCD2, and the upregulation of RCD1 and RCD2 can in turn induce the expression of disease resistance-related genes in rice.

[0115] (3) Co-expression of RCD1 and RCD2 conferred resistance to bacterial blight in rice.

[0116] Experimental methods: The bacterial suspension for bacterial blight infection was prepared using the same method as in Example 3S2(1). The bacterial suspension was applied to rice flag leaves using a leaf-cutting method (cutting point: approximately 1.5 cm from the leaf tip towards the stem). Samples were taken at 10 dpi, and the length of bacterial blight lesions and biomass were measured. The results are shown in […]. Figure 5 (exist Figure 5 The top image shows the condition of leaves after infection with white leaf blight, and the bottom image is a statistical chart of lesion length. RCD12 represents a stable T2 generation rice line containing both RCD1 and RCD2. RC D12 Native represents a negative progeny line of the T2 generation isolated from the T1 generation RCD12 heterozygous line.

[0117] The separation method is as follows: T1 generation rice seeds are germinated, and each rice plant is subjected to PCR testing. Lines containing RCD1 and RCD2 are positive lines, and lines not containing RCD1 and RCD2 are negative lines.

[0118] Depend on Figure 5It can be seen that the average lesion lengths of WT, RCD12, and RCD12 Native were 151.27 mm, 12.06 mm, and 141.21 mm, respectively. This indicates that the lesion length and biomass of the transformed lines simultaneously expressing RCD1 and RCD2 were significantly smaller than those of the negative-positive lines and WT lines isolated from the RCD1+2 progeny. Therefore, co-expression of RCD1 and RCD2 does indeed confer resistance to bacterial blight in rice.

[0119] In summary, by adopting the technical solution provided by this invention, the introduction of exogenous RCD1 and RCD2 genes into tobacco is beneficial to inducing tobacco hypersensitivity (HR) response and reactive oxygen species (ROS) burst, thereby endowing tobacco with resistance to Phytophthora capsulatum. The introduction of exogenous RCD1 and RCD2 genes into rice improves the rice's resistance to bacterial blight without affecting the rice's agronomic traits.

[0120] 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. A combination of genes for use in improving the disease resistance of plants and / or in breeding disease-resistant plants; the combination of genes comprising an RCD1 gene and an RCD2 gene; the nucleotide sequence of the RCD1 gene is shown as SEQ ID NO. 1; the nucleotide sequence of the RCD2 gene is shown as SEQ ID NO. 2; the disease resistance comprises resistance to Phytophthora capsici in tobacco and / or resistance to Xanthomonas oryzae in rice; the plants comprise tobacco and / or rice.

2. A combination of proteins for use in improving the disease resistance of plants and / or in breeding disease-resistant plants, the combination of proteins being encoded by the combination of genes of claim 1, the combination of proteins comprising an RCD1 protein and an RCD2 protein; the amino acid sequence of the RCD1 protein is shown as SEQ ID NO. 3; the amino acid sequence of the RCD2 protein is shown as SEQ ID NO. 4; the disease resistance comprises resistance to Phytophthora capsici in tobacco and / or resistance to Xanthomonas oryzae in rice; the plants comprise tobacco and / or rice.

3. A biological material comprising the combination of genes of claim 1 for use in improving the disease resistance of plants and / or in breeding disease-resistant plants, the biological material comprising a recombinant vector comprising the combination of genes of claim 1 or an engineered bacterium comprising the recombinant vector; the disease resistance comprises resistance to Phytophthora capsici in tobacco and / or resistance to Xanthomonas oryzae in rice; the plants comprise tobacco and / or rice.

4. The recombinant vector of claim 3, further comprising a promoter; the promoter is Xa23-pro.

5. The engineered bacterium of claim 3, further comprising a promoter; the promoter is Xa23-pro.

6. A method for improving the disease resistance of plants and / or for breeding disease-resistant plants, comprising introducing the combination of genes of claim 1 into target plants; the disease resistance comprises resistance to Phytophthora capsici in tobacco and / or resistance to Xanthomonas oryzae in rice; the plants comprise tobacco and / or rice.

7. The method of claim 6, wherein the introducing comprises injecting a transformation solution comprising an RCD1 gene and an RCD2 gene expression vector into target plants. ​ ​ ​ 4. Use according to claim 3, characterized in that, ​ 5. A method for enhancing disease resistance in plants and / or breeding disease resistant plants, characterized in that, ​ ​ 6. The method of claim 5, wherein, ​

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