Nikolajeva broad-spectrum disease-resistant gene rpl-avh23 and the encoded protein and application thereof

Abstract

The application discloses a broad-spectrum disease-resistant gene Rpl-avh23 of Nicotiana plumbaginifolia, and an encoding protein and application thereof. The application separates a disease-resistant gene Rpl-avh23 from the Nicotiana plumbaginifolia, the full-length cDNA of the gene is 2154 bp, the nucleotide sequence is shown as SEQ ID NO:1; the protein encoded by the gene is an intracellular immune receptor protein with a full length of 717 amino acids, and the amino acid sequence is shown as SEQ ID NO:2. The application finds that the Rpl-avh23 gene can improve the resistance of plants to oomycetes, fungi and / or viruses through transcription mode analysis and plant expression analysis, and therefore, the disease-resistant gene Rpl-avh23 can be applied to improving the disease resistance of plants or plant breeding.

Description

Technical Field

[0001] This invention belongs to the field of molecular plant pathology technology, and specifically relates to a broad-spectrum disease resistance gene Rpl-avh23 in Nicotiana benthamiana, its encoded protein, and its applications. Background Technology

[0002] Nicotiana benthamiana is an annual herbaceous plant belonging to the genus Nicotiana in the family Solanaceae, native to Australia. As an internationally recognized important model plant, it is the preferred host system for studying plant-pathogen interactions, identifying resistance genes (R genes), and effector proteins due to its high sensitivity to various plant pathogens (including viruses, bacteria, fungi, and oomycetes). During infection, pathogens secrete a variety of virulence molecules to disrupt the physical defenses and immune system of plant cells. Among these, effector proteins are numerous and have diverse attack sites, playing a crucial role throughout the entire pathogen infection process. Most R genes encode immune receptors (NLRs) containing nucleotide-binding domains (NB) and leucine-rich repeat regions (LRR). These receptors recognize effector proteins secreted by pathogens and rapidly transmit the recognition signal to downstream kinases and signal transduction networks, triggering a rapid and intense immune response, including reactive oxygen species bursts, cell wall reinforcement, and hypersensitivity (HR) reactions, thereby limiting the spread of pathogens.

[0003] Given the central role of Nicotiana benthamiana in elucidating plant immune mechanisms, the efficient discovery, verification, and utilization of disease resistance genes (including NLR genes from Nicotiana benthamiana itself and its homologous crops) using the Nicotiana benthamiana system has become an important strategy for modern plant disease control. Through the transient expression system of Nicotiana benthamiana, researchers can rapidly identify the functional specificity of candidate NLR genes and elucidate their interaction patterns with effector proteins, thus providing crucial gene resources and theoretical basis for breeding new crop varieties with broad-spectrum and durable resistance. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide a protein encoded by the broad-spectrum disease resistance gene Rpl-avh23 in Nicotiana benthamiana.

[0005] Another objective of this invention is to provide a broad-spectrum disease resistance gene Rpl-avh23 for Nicotiana benthamiana.

[0006] Another object of the present invention is to provide the application of the aforementioned broad-spectrum disease resistance gene Rpl-avh23 in Nicotiana benthamiana and its encoded protein.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] The protein encoded by the broad-spectrum disease resistance gene Rpl-avh23 in Nicotiana benthamiana has the amino acid sequence shown in SEQ ID NO: 2.

[0009] The protein encoded by the broad-spectrum disease resistance gene Rpl-avh23 in Nicotiana benthamiana is an intracellular immune receptor protein, comprising two parts: the NB-ARC functional domain and the LRR functional domain.

[0010] The broad-spectrum disease resistance gene Rpl-avh23 in Nicotiana benthamiana has the nucleotide sequence shown in SEQ ID NO: 1.

[0011] The biological materials related to the protein encoded by the broad-spectrum disease resistance gene Rpl-avh23 of Nicotiana benthamiana mentioned above are any one or more combinations of the following biological materials:

[0012] (1) Expression cassette containing the above-mentioned gene Rpl-avh23;

[0013] (2) Recombinant vectors containing the above-mentioned gene Rpl-avh23;

[0014] (3) A recombinant vector containing the expression cassette described in (1);

[0015] (4) Transformed bacteria containing the above-mentioned gene Rpl-avh23;

[0016] (5) Transforming bacteria containing the expression cassette described in (1);

[0017] (6) Transformation bacteria containing the recombinant vector described in (2) or (3).

[0018] Furthermore, the starting vector of the recombinant vector can be any binary vector that can be used for Agrobacterium-mediated transformation of plants or a vector that can be used for plant micro-projectile attack, such as the pBin series vectors. The present invention uses the overexpression vector pBin series vectors.

[0019] Furthermore, the originating strain of the transforming bacteria is Agrobacterium; preferably Agrobacterium GV3101.

[0020] The application of the broad-spectrum disease resistance gene Rpl-avh23 of Nicotiana benthamiana, its encoded protein, or biological materials in improving plant disease resistance.

[0021] The application of the broad-spectrum disease resistance gene Rpl-avh23 of Nicotiana benthamiana, its encoded protein, or biological material in improving plant disease resistance is to enhance plant resistance to fungi, oomycetes, and / or viruses by overexpressing the broad-spectrum disease resistance gene Rpl-avh23 of Nicotiana benthamiana.

[0022] The plants mentioned include Nicotiana benthamiana.

[0023] The disease resistance mentioned refers to resistance to oomycetes, fungi, and viruses.

[0024] The oomycetes include Phytophthora; further include Phytophthora capsici.

[0025] The fungi mentioned include anthrax fungi; further including fruit anthrax fungi.

[0026] The viruses mentioned include tobacco viruses; further including tobacco mosaic virus.

[0027] The application of the broad-spectrum disease resistance gene Rpl-avh23 of Nicotiana benthamiana, its encoded protein, or biological materials in the cultivation of disease-resistant plants.

[0028] The application of the broad-spectrum disease resistance gene Rpl-avh23 of Nicotiana benthamiana, its encoded protein, or biological materials in the cultivation of disease-resistant plants is to improve the plant's resistance to fungi, oomycetes, and / or viruses by overexpressing the broad-spectrum disease resistance gene Rpl-avh23 of Nicotiana benthamiana.

[0029] The plant in question is Nicotiana benthamiana.

[0030] The disease resistance mentioned refers to resistance to oomycetes, fungi, and viruses.

[0031] The oomycetes include Phytophthora; further include Phytophthora capsici.

[0032] The fungi mentioned include anthrax fungi; further including fruit anthrax fungi.

[0033] The viruses mentioned include tobacco viruses; further including tobacco mosaic virus.

[0034] The present invention has the following advantages and effects compared with the prior art:

[0035] (1) The broad-spectrum disease resistance gene Rpl-avh23 of Nicotiana benthamiana in this invention was isolated from Nicotiana benthamiana. The full-length cDNA of this gene is 2154 bp (SEQ ID NO: 1). The protein encoded by this gene is an intracellular immune receptor protein composed of 717 amino acids (SEQ ID NO: 2). Through plant expression analysis, it was found that Rpl-avh23 can improve the plant's resistance to oomycetes, fungi and / or viruses. Therefore, this disease resistance gene Rpl-avh23 can be applied to plant disease resistance breeding and the synthesis of new disease resistance genes, and is a disease resistance material with great application value.

[0036] (2) This invention explores that transient expression of the broad-spectrum disease resistance gene Rpl-avh23 in Nicotiana benthamiana can cause an allergic necrosis reaction after 48 hours.

[0037] (3) In this invention, the broad-spectrum disease resistance gene Rpl-avh23 of Nicotiana benthamiana was transiently expressed in Nicotiana benthamiana. After 48 hours, it was inoculated with Phytophthora capsici, Anthracnose cylindrica and Nicotiana mosaic virus. It was observed that the lesion area after Rpl-avh23 expression was significantly reduced compared with the negative control, and the virus content was significantly reduced. The results showed statistical differences.

[0038] (4) In this invention, the broad-spectrum disease resistance gene Rpl-avh23 of Nicotiana benthamiana was transiently expressed in Nicotiana benthamiana. After 48 hours, RNA was extracted and reverse transcribed into cDNA. The expression level of tobacco resistance genes was measured by real-time PCR. The results showed that after overexpression of Rpl-avh23, the expression levels of genes such as NbPR1, NbPR2, NbLOX, and NbHir1 increased accordingly. Attached Figure Description

[0039] Figure 1 This is a diagram showing the phytosensitive necrosis response induced by transient expression of Rpl-avh23 in tobacco leaves.

[0040] Figure 2 The graph shows the effects of Rpl-avh23 and GFP expression on tobacco infection by Phytophthora capsici (A) and the statistical analysis of leaf lesion area (B). In A: the left side of the leaf is after transient GFP expression followed by Phytophthora capsici inoculation; the right side of the leaf is after transient Rpl-avh23 expression followed by Phytophthora capsici inoculation.

[0041] Figure 3 The graph shows the effects of Rpl-avh23 and GFP expression on the infection of *Nicotiana benthamiana* by *Anthracis rubra* (A) and the statistical analysis of leaf lesion area (B). In A: the left side of the leaf is inoculated with *Anthracis rubra* after transient GFP expression; the right side of the leaf is inoculated with *Anthracis rubra* after transient Rpl-avh23 expression.

[0042] Figure 4 The figures (A) show the effect of Rpl-avh23 and GFP expression on TMV infection of Nicotiana benthamiana and (B) show the TMV biomass statistical analysis. In A, the left side of the leaf shows the TMV inoculation after transient GFP expression; the right side of the leaf shows the TMV inoculation after transient Rpl-avh23 expression.

[0043] Figure 5 This is a diagram showing the transient expression of the Rpl-avh23-activated resistance gene in Nicotiana benthamiana. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0045] The *Phytophthora capsici* involved in the embodiments of this invention is a conventional *Phytophthora*, which can be obtained through commercial means or isolated from nature.

[0046] The fruit anthracnose fungus involved in the embodiments of the present invention is a conventional pathogenic fungus that can be obtained through commercial means or isolated from nature.

[0047] The tobacco mosaic virus involved in the embodiments of the present invention is a conventional plant virus that can be obtained through commercial means or isolated in nature.

[0048] The pBin vector and Agrobacterium GV3101 involved in the embodiments of the present invention can be obtained through conventional commercial means.

[0049] Example 1: Cloning and Acquisition of the Rpl-avh23 Gene

[0050] (1) Based on the analysis of the Nicotiana benthamiana genome, it was predicted that Nicotiana benthamiana may encode the Rpl-avh23 gene. We designed primers (upstream primer Rpl-avh23-F and downstream primer Rpl-avh23-R) and successfully cloned the Rpl-avh23 gene from Nicotiana benthamiana cDNA. Its nucleotide sequence is shown in SEQ ID NO: 1, and its encoded protein amino acid sequence is shown in SEQ ID NO: 2.

[0051] Rpl-avh23 gene (SEQ ID NO: 1, length 2154bp):

[0052]

[0053] Rpl-avh23 protein (SEQ ID NO: 2, length 717 aa):

[0054] MVGFDRETEVLMEKLVTGPRQLDVISIFGMPGLGKTTLAKRLYDHEAISNRFDIRLWCCSSQSYDKRSLLFELLNHICKYDTSTKEKSDDELAEMLYKYLKGKRYLIVVDDVWSQEAWDDIIRSFPDDDTGSRIILTSRLESIATYAMIDSSPHFLRMFTEEESWMLMKQKVFKEDNCYCPQQLEEIGKQIASKCGGLPLAVILVAGLLAKHDKEVVYWQEVGESLKSKIQGCMDIVESSYQHLPIHLQKCFLYFASFLEDQKVPVQKLIRLWIAENFVEASSTLKSLEMVAMDYLMDLISSNLVMVAKINSLGELKSIHIHDLIREFSLVKAKNENFLLRINRIGEFCSSRGSRLRRGLGIFPSPESCCKRVNILAPSELLLGERFRLGQNINTLRFLPGVYSPIVDYDLTWKHLRVLDLGSVRLRQTLVDALQHLIHLKYLELQLYFHNIPSSVCNLKKLETFIVFGYHDQTCIPITIWNMKRLRHLHISPLYTNKLLHSLDNLQTCSDLVVSPGMYYRNFMKRFSNLEKLSCQLYGSGTTSGNFFPAFETLGKLKSLKIGVSGAVVDPYGFENFTYPSNLKKLTLVYLRLPWSKLSTIGTLSNLEVLKLEESSFIGRKWDVNDGEFPTLKVLKLKKLGFSEWTASDDSYPNLQKVLVHSCWSLEEIPECFGSNCTLQLIELRSCGDDVVNSALKIKEMQIEEMGNSEFKVIISK.

[0055] Forward primer Rpl-avh23-F:

[0056] 5'-CTGTACAAGGGTACCCCCGGGATGGTTGGCTTTGATAGAGAG-3';

[0057] Reverse primer Rpl-avh23-R:

[0058] 5'-AGAGGATCCGTCGACCCCGGGTCACTTTGAAATAATGACTTTGAATTC-3'.

[0059] (2) Total RNA (ribonucleic acid) was extracted from tobacco leaves (Sangon Biotech kit). Then, the mRNA was transcribed into cDNA by reverse PCR (5*Prime ScriptRT MaterMix amplification reagent). The nucleotide sequence shown in SEQ ID NO: 1 was amplified from the tobacco cDNA by PCR using the above upstream and downstream primers. The PCR program was as follows: 95℃, 3min; 95℃, 15sec; 52℃, 15sec; 72℃, 90sec; 36 cycles; 72℃, 5min.

[0060] (3) The PCR products were purified and separated by electrophoresis on a 1% agarose gel. The agarose gel containing the target fragment was cut and collected in a 2ml centrifuge tube, and recovered using the Omega Gel Extraction Kit. The gene fragment with sequence SEQ ID NO: 1 was obtained by sequencing verification.

[0061] Example 2: Transient expression of Rpl-avh23 in Benedict's smoke

[0062] Construction of a transient expression vector for Bunsen's smoke:

[0063] 1. Construction of transient expression vector for Nicotiana benthamiana: The amplified Rpl-avh23 target fragment was ligated to the pBin vector using a homologous recombinase (linkage site: SmaI) to obtain the recombinant plasmid pBin::Rpl-avh23 containing the sequence of SEQ ID NO: 1; The GFP sequence (SEQ ID NO: 3, the amino acid sequence of the protein it encodes is shown in SEQ ID NO: 4) was ligated to the pBin vector using a homologous recombinase (linkage site: SmaI) to obtain the recombinant plasmid pBin::GFP containing GFP green fluorescent protein. After propagation in Escherichia coli DH5α strain, the plasmids were extracted, and the recombinant plasmid pBin::GFP and the recombinant plasmid pBin::Rpl-avh23 containing the sequence of SEQ ID NO: 1 were respectively transformed into Agrobacterium GV3101.

[0064] GFP gene (SEQ ID NO: 3, length 720bp):

[0065] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTGA。

[0066] GFP protein (SEQ ID NO: 4, length 239 aa)

[0067] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK.

[0068] 2. Transient expression of *Nicotiana benthamiana*: Single colonies of *Agrobacterium* containing recombinant plasmids pBin::GFP and pBin::Rpl-avh23 were picked and placed into 2 mL of LB broth containing kanamycin (50 μg / mL), and incubated at 28℃ and 180 r·min. -1 Culture for 1–2 days; use Agrobacterium containing the GFP control vector as a control. 4000 r·min -1 Collect bacterial cells by centrifugation for 4 min, gently blow the bacterial precipitate with pre-cooled MgCl2, and then centrifuge at 4000 r·min. -1 Centrifuge for 4 min, repeat three times. Adjust the OD of the bacterial culture with MgCl2. 600 After the value was 0.05 to 0.1, tobacco was injected separately, with each of the two types of Agrobacterium injected into half of a Bunsenior tobacco leaf.

[0069] 3. Results are as follows Figure 1 As shown, after 48 hours, the injected concentration was OD 600 Necrosis appeared in tobacco leaves with a concentration of 0.1. GFP served as a negative control, while INF1, a classic pathogenic Phytophthora elicitor that induces hypersensitive necrosis in plant cells, served as a positive control.

[0070] Example 3: Phytophthora capsici infecting tobacco leaves

[0071] 1. Culture of Phytophthora capsici

[0072] Strain: Phytophthora capsici is a common Phytophthora fungus that can be obtained commercially or isolated in nature.

[0073] Carrot agar (CA) (1L): Juice 300g of carrots, filter through gauze, and sterilize at 121℃ for 20min. Add 1.5% (w / v) agar powder to the solid medium.

[0074] The mycelial blocks of Phytophthora capsici strain preserved in the laboratory were inoculated onto carrot culture medium and cultured at 28°C for 5 days.

[0075] 2. Infection Experiment

[0076] Forty-eight hours after injecting Agrobacterium tumefaciens into Nicotiana benthamiana, Phytophthora capsici mycelium cakes were punched out using a puncher and inoculated onto the underside of the tobacco leaves after transient expression, with the mycelial side facing down. One mycelium cake was inoculated at the same position on each side of the leaf and placed in a plastic box to keep it moist.

[0077] Two days later, the size of the lesions infected with Phytophthora capsici was observed under ultraviolet light. The results were as follows: Figure 2 A and Figure 2 As shown in B: When the tobacco disease resistance-related gene Rpl-avh23 was transiently expressed in tobacco, and then inoculated with Phytophthora capsici 48 hours later, it was observed that the lesions after Rpl-avh23 expression were significantly smaller than those in the negative control, and biostatistical analysis showed that there was a significant difference in the lesion area between the two treatments.

[0078] Example 4: Fruit-borne anthracnose fungus infects tobacco leaves

[0079] 1. Culture of *Anthracnose spp.*

[0080] Strain: Colletotrichum fructicola is a common anthrax bacterium that can be obtained commercially or isolated in nature.

[0081] The mycelial blocks of the fruit-borne anthracnose strain preserved in the laboratory were inoculated onto PDA medium and cultured in a culture room at 28°C for 5 days.

[0082] 2. Infection Experiment

[0083] Forty-eight hours after transient expression in *Tobacco Benedict*, fruit anthracnose fungal cakes were punched out using a puncher. The mycelial side was inoculated onto the underside of the leaves after transient expression in tobacco, with one cake inoculated at the same position on each side of the leaf. The cakes were then placed in a plastic box to maintain humidity.

[0084] Two days later, the size of the lesions infected with *Anthracnose* on the fruit was observed under ultraviolet light. The results were as follows: Figure 3 A and Figure 3 As shown in B: When the tobacco disease resistance-related gene Rpl-avh23 was transiently expressed in tobacco, and then inoculated with fruit anthracnose 48 hours later, it was observed that the lesions after Rpl-avh23 expression were significantly smaller than those in the negative control, and biostatistical analysis showed that there was a significant difference in the lesion area between the two treatments.

[0085] Example 5: Tobacco mosaic virus infects Benedictine tobacco leaves

[0086] Forty-eight hours after transient expression in *N. benthamiana*, 100 mg of leaves infected with tobacco mosaic virus (TMV) were homogenized in 1 mL of 50 mM sodium phosphate buffer (pH 7.0), centrifuged at 5000 × g for 3 minutes, and the supernatant was collected. Before inoculation, 10 μL of the virus homogenate and a small amount of quartz sand were added to the leaf, and the entire leaf was gently rubbed by hand. Results are as follows: Figure 4 A and Figure 4 As shown in B: After 7 days, wilting symptoms were significantly reduced compared to the negative control after Rpl-avh23 expression. The biomass of tobacco mosaic virus was significantly reduced by qPCR. Primer information for quantitative real-time PCR is shown in Table 1.

[0087] Table 1 Primer information for quantitative real-time PCR

[0088]

[0089] The RT-PCR reaction procedure is as follows: 95℃ pre-denaturation for 30s; 40 cycles; 95℃ denaturation for 30s; 60℃ annealing and extension for 30s; 40 cycles.

[0090] After cycling, melting curves were plotted: fluorescence signals were collected every 1℃ from 60℃ to 95℃. Quantitative RT-PCR analysis was then performed using an RT-PCR instrument.

[0091] Example 6: Rpl-avh23 expression induces upregulation of transcriptional levels of tobacco Benedict's resistance genes

[0092] 1. RNA extraction from tobacco leaves

[0093] Total RNA was extracted from tobacco leaves using a Sangon Biotech kit. cDNA was synthesized using 5*Prime ScriptRT MaterMix amplification reagent.

[0094] 2. The expression level of tobacco resistance gene was detected by RT-PCR. The primer information for real-time PCR is shown in Table 2.

[0095] Table 2 Primer Information for Quantitative Real-Time PCR

[0096]

[0097] The RT-PCR reaction procedure is as follows: 95℃ pre-denaturation for 30s; 40 cycles; 95℃ denaturation for 30s; 60℃ annealing and extension for 30s; 40 cycles.

[0098] Melting curves were plotted after cycling: fluorescence signals were collected at 1℃ every 1℃ from 60℃ to 95℃. Quantitative RT-PCR analysis was performed using an RT-PCR instrument to analyze the expression of Rpl-avh23 at different time points after infection.

[0099] 3. Results:

[0100] The results are as follows Figure 5 As shown, transient expression of Rpl-avh23 in tobacco leaves enhanced the expression of tobacco resistance-related genes, with the enhancement reaching several times; this change demonstrates its important role in the broad-spectrum disease resistance of tobacco.

[0101] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The protein encoded by the broad-spectrum disease resistance gene Rpl-avh23 in Nicotiana benthamiana, characterized by: The amino acid sequence of the encoded protein is shown in SEQ ID NO:

2.

2. The broad-spectrum resistance gene Rpl-avh23 of Nicotiana benthamiana as described in claim 1.

3. The broad-spectrum disease resistance gene Rpl-avh23 for Nicotiana benthamiana according to claim 2, characterized in that: Its nucleotide sequence is shown in SEQ ID NO:

1.

4. The biomaterials related to the protein encoding the broad-spectrum disease resistance gene Rpl-avh23 of Nicotiana benthamiana as described in claim 2, characterized in that: It can be any one or more combinations of the following biological materials: (1) An expression cassette containing the gene Rpl-avh23 as described in claim 2 or 3; (2) A recombinant vector containing the gene Rpl-avh23 as described in claim 2 or 3; (3) A recombinant vector containing the expression cassette described in (1); (4) Transformed bacteria containing the gene Rpl-avh23 as described in claim 2 or 3; (5) Transforming bacteria containing the expression cassette described in (1); (6) Transformation bacteria containing the recombinant vector described in (2) or (3).

5. The related biomaterial according to claim 4, characterized in that: The starting vectors for the recombinant vectors include the pBin series vectors; And / or, the originating strain of the transforming bacteria is Agrobacterium.

6. The application of the encoded protein of claim 1, the broad-spectrum resistance gene Rpl-avh23 of Nicotiana benthamiana according to any one of claims 2-3, or the related biological material according to any one of claims 4-5, characterized in that: For any one or more of the following applications: (a) Applications in improving plant disease resistance; (b) Application in the cultivation of disease-resistant plants.

7. The application according to claim 6, characterized in that: The plant in question is Nicotiana benthamiana.

8. The application according to claim 6 or 7, characterized in that: Overexpression of the broad-spectrum disease resistance gene Rpl-avh23 in Nicotiana benthamiana enhances plant resistance to oomycetes, fungi, and / or viruses.

9. The application according to claim 8, characterized in that: The oomycetes mentioned include Phytophthora; The fungi mentioned include anthrax bacteria; The viruses mentioned include tobacco viruses.

10. The application according to claim 9, characterized in that: The aforementioned Phytophthora includes Phytophthora capsici; The anthrax bacteria mentioned include *Anthrax spp.* The tobacco viruses mentioned include tobacco mosaic virus.

Images