A plant susceptibility gene SNIPER and its application

Abstract

This invention provides the application of the plant susceptibility gene SNIPER in regulating plant disease resistance, belonging to the fields of plant molecular biology and plant genetic engineering. This invention relates to a plant disease susceptibility gene SNIPER, its recombinant silencing vector, and its applications. This gene is derived from *Nicotiana benthamiana* and has the nucleotide sequence shown in SEQ ID NO.1, with the encoded product having the amino acid sequence shown in SEQ ID NO.3. This invention can induce resistance to multiple pathogens in tobacco by inhibiting the expression of this gene, which is of great significance in improving disease resistance in crop breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology and plant genetic engineering. Specifically, this invention relates to the application of the plant susceptibility gene SNIPER in regulating plant disease resistance, and a method for constructing a plant disease resistance model by silencing the SNIPER gene. Background Technology

[0002] Phytophthora blight, caused by Phytophthora, is one of the most devastating diseases in agricultural production, causing enormous economic losses globally each year and seriously threatening global food security. To this day, late blight caused by Phytophthora blight continues to restrict the development of potato and tomato production. [1] In addition, *Phytophthora capsici* and *Phytophthora parasitica* can cause serious damage to various Solanaceae plants, including peppers and tobacco. [2] Phytophthora is extremely difficult to control due to its rapid onset of disease, high prevalence, wide distribution, and strong adaptability to hosts in the field. Therefore, discovering and utilizing new genes to improve plant resistance to Phytophthora is of great significance for improving plant resistance.

[0003] [1] Fry, W. 2008. Phytophthora infestans: the plant (and R gene) destroyer. Molecular Plant Pathology, 9(3): 385-402.

[0004] [2] Lamour, KH, Stam R., Jupe, J., and Huitema E. 2012. The oomycete broad-host-range pathogen Phytophthora capsici. Molecular Plant Pathology, 13(4): 329-337. Summary of the Invention

[0005] One of the objectives of this invention is to provide a gene SNIPER.

[0006] The second objective of this invention is to provide applications for the gene SNIPER.

[0007] The specific details of this invention are as follows:

[0008] This invention provides a gene SNIPER, which is derived from tobacco and has the nucleotide sequence shown in SEQ ID NO.1.

[0009] The present invention also provides a protein encoded by the gene SNIPER, or a protein having an amino acid sequence with at least 50% similarity to the protein encoded by the gene SNIPER. The amino acid sequence of the protein encoded by the gene SNIPER described in this invention is shown in SEQ ID NO.3, and the amino acid sequence of the protein having at least 50% similarity to the protein encoded by the gene SNIPER is shown in SEQ ID NO.4.

[0010] The present invention also provides a recombinant expression vector comprising the aforementioned gene SNIPER.

[0011] The expression vector is preferably a plant transformation plasmid, such as pBin, pCambia, or pTF101.1.

[0012] Preferably, the recombinant expression vector is pBin:SNIPER-HA, obtained by inserting the gene SNIPER into the binary vector pBin:HA restriction site SmallI containing C-terminal HA.

[0013] The present invention also provides a silencing fragment of the silencing gene SNIPER, the nucleotide sequence of which is SEQ ID NO.2.

[0014] The present invention also provides a gene silencing vector for the silencing gene SNIPER, and in some specific instances, a gene silencing vector comprising a silencing fragment of the silencing gene SNIPER.

[0015] The gene silencing vector is preferably a plant transformation plasmid, which can be the silencing vector pTRV2.

[0016] Preferably, the recombinant silencing vector is the vector pTRV2:SNIPER obtained by inserting the silencing fragment of the silencing gene SNIPER into the PstI restriction site of the viral silencing vector pTRV2.

[0017] The present invention also provides a transformant obtained by introducing the gene silencing vector into a host cell, wherein the host cell is preferably an Escherichia coli cell or an Agrobacterium cell.

[0018] The present invention also provides the application of the gene SNIPER or its encoded protein or silencing fragment, or gene silencing vector or transformant, in improving plant immune resistance or disease resistance. Preferably, the application is to silence or interfere with the expression of the gene SNIPER.

[0019] The present invention also provides the application of the gene SNIPER or its encoded protein or silencing fragment, or gene silencing vector or transformant in enhancing the immune resistance of plants to pathogens or enhancing plant resistance to diseases caused by pathogens. Preferably, the application is to silence or interfere with the expression of the gene SNIPER.

[0020] The present invention also provides the application of the gene SNIPER or its encoded protein or silencing fragment, or gene silencing vector or transformant in the breeding of plants with immune resistance to pathogenic bacteria, preferably, the application is to silence or interfere with the expression of the gene SNIPER.

[0021] The present invention also provides the application of the tobacco gene SNIPER or its encoded protein or silencing fragment, or gene silencing vector or transformant, in obtaining significant disease resistance after introduction into plants.

[0022] The plant described in this invention may be selected from tobacco, tomato, potato or soybean.

[0023] The pathogenic bacteria described in this invention are Phytophthora indicum or Pseudomonas syringae, such as Phytophthora indicum P26 and the mutant strain Pto DC3000ΔhopQ1-1 of Pseudomonas syringae.

[0024] We constructed overexpression and RNA interference vectors using genetic engineering techniques. In transiently overexpressed tobacco leaves, we found that SNIPER negatively regulates the resistance of *Nicotiana benthamiana* to *Phytophthora* and other bacteria. Using virus-induced gene silencing (VIGS), we silenced the SNIPER gene in *Nicotiana benthamiana*. Silenced SNIPER significantly enhanced the activation of the plant's innate immune response and strengthened resistance to *Phytophthora* and other bacteria, indicating that the SNIPER protein has a disease-susceptibility function. Reducing the expression levels of the SNIPER gene and protein will play a crucial role in tobacco disease resistance. Furthermore, as an important economic crop and a typical representative of the Solanaceae family, research on SNIPER in tobacco can drive related research in many other plants such as tomatoes and potatoes. These studies will better elucidate the disease resistance functions of plants against *Phytophthora* and different pathogens, providing excellent disease-resistant gene resources for disease-resistant genetic engineering breeding.

[0025] Beneficial effects of the present invention

[0026] The protein encoded by the SNIPER gene described in this invention acts as a susceptibility factor, suppressing the plant's innate immunity. Therefore, rationally reducing the expression level of SNIPER will benefit the enhancement of plant disease resistance. Silencing the SNIPER gene in plants does not affect plant growth traits but can significantly enhance plant resistance to Phytophthora and bacteria. This invention can be applied to crop breeding for disease resistance improvement, and is expected to improve plant resistance to Phytophthora blight, thereby achieving the goal of increasing yield and reducing pesticide use. Attached Figure Description

[0027] Figure 1 TRV:SNIPER SNIPER gene expression detection in silent tobacco. Real-time quantitative PCR was used to detect the SNIPER gene expression level in silent tobacco, where TRV:GFP represented the control plant, and -1 and -2 represented different strains.

[0028] Figure 2 TRV:SNIPER silences tobacco without affecting plant growth traits, where TRV:GFP, -1, and -2 represent different strains.

[0029] Figure 3 The treatment of tobacco with TRV:GFP and TRV:SNIPER resulted in disease symptoms after inoculation with Phytophthora nicotineae, with TRV:GFP, -1, and -2 being different strains.

[0030] Figure 4 Expression of pBin:eGFP-HA and pBin:SNIPER-HA transgenic tobacco proteins was detected. Western blot analysis was performed to detect the expression levels of control eGFP-HA and SNIPER-HA, using anti-HA as the detection antibody.

[0031] Figure 5 Symptoms of disease observed 3 days after inoculation of pBin:eGFP-HA and pBin:SNIPER-HA transgenic tobacco with Phytophthora infestans.

[0032] Figure 6 The number of colonies in tobacco treated with TRV:GFP and TRV:SNIPER after inoculation with Pto DC3000ΔhopQ1-1 bacteria was statistically analyzed. TRV:GFP, -1, and -2 represent different strains. Detailed Implementation

[0033] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. The primers involved in the embodiments of the present invention were synthesized by Shanghai Sangon Biotech Co., Ltd. The mutant strains Pto DC3000ΔhopQ1-1 of Phytophthora tobaccois P26 and Pseudomonas syringae are strains from our laboratory, and our laboratory promises to permanently make them available to the public.

[0034] Example 1. Cloning and sequence structure analysis of the SNIPER gene

[0035] Tobacco (Nicotiana benthamiana) seeds were sown directly in pots filled with nutrient soil and cultured in a greenhouse (21-23℃, 14h light / 10h darkness). Six-week-old plants were used for RNA extraction.

[0036] Total RNA extraction: Using tobacco leaves as material, total RNA was extracted using the Omega RNA extraction kit according to the instructions, and the RNA content and quality were detected using a spectrophotometer.

[0037] First-strand reverse transcription: Using 0.7 μg of RNA as a template, cDNA synthesis was performed according to the instructions for use of Takara's PrimeScript reverse transcriptase reagent, and the volume was adjusted to 20 μL. An appropriate amount of the reverse transcription product was used for subsequent gene cloning PCR.

[0038] Using the first strand of cDNA as an RT-PCR template, PCR was performed using standard methods to amplify the SNIPER gene (SEQ ID NO.1) fragment or the full-length gene:

[0039] PCR amplification primer sequences:

[0040] Upstream primer:

[0041] (5'-GATAGCCGGTACCCCATGGCTTCAGAATCAGAATCAGC-3')

[0042] Downstream primer:

[0043] (5'-CATCGTATGGGTACCCAAGCTCTGGTTCATGATCATGTTC-3'),

[0044] The 50 μL reaction mixture consisted of 25 μL 2×Phanta Max Master Mix, 1 μL template cDNA, and water added to a final volume of 50 μL. The PCR amplification program was: 95°C pre-denaturation for 5 minutes, 95°C denaturation for 30 seconds, 57°C annealing for 30 seconds, 72°C extension for 30 seconds, repeated 35 times, followed by a final extension at 72°C for 10 minutes. Electrophoresis was performed on an agarose gel, followed by ethidium bromide (EB) staining and photographing. The results were recorded, and the SNIPER PCR products were recovered by gel excision. The electrophoretic bands were recovered using the Agarose Gel DNA PCR Kit (TaKaRa). The PCR product of SNIPER recovered from gel excision was ligated into the smallI-digested pBin::HA vector using the ClonExpress II One Step Cloning Kit (Vazyme) according to the instructions to obtain the pBin::SNIPER-HA plasmid (containing the sequence SEQ ID NO.1). Simultaneously, eGFP was ligated as a control gene to obtain the pBin::eGFP-HA plasmid (containing the sequence SEQ ID NO.11). Both plasmids were transformed into E. coli competent cells JM109, plated on LB agar plates (containing 50 μg / mL kanamycin), and incubated at 37°C for 16 hours. After colony PCR verification, three clones were picked, and plasmids were extracted using a plasmid extraction kit (Takara) and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequences are shown in SEQ ID NO.1 and SEQ ID NO.11. The correctly sequenced plasmid was electroporated into Agrobacterium GV3101, plated on LB agar plates (containing 50 μg / mL kanamycin and 50 μg / mL rifampin), and incubated at 30°C for 48 hours. After colony PCR verification, the correct clones were picked for subsequent experiments.

[0045] Example 2. Construction of the silencing vector TRV2:SNIPER:

[0046] Total RNA was extracted from tobacco and cDNA was synthesized using reverse transcription. Primers were designed based on the SNIPER sequence (SEQ ID NO.1) to amplify a partial fragment for the construction of the SNIPER gene silencing vector.

[0047] Upstream primer:

[0048] 5'-CGACAAGACCCTGCAACCCTTTATTTTAGCCTCTTTCACAAAAC-3'

[0049] Downstream primer:

[0050] 5'-GAGAAGAGCCCTGCACTTTTCAATACAATTCCCATGAAACCT-3'

[0051] The fragment in SEQ ID NO.1 was amplified by PCR to obtain a partial gene sequence of 388 bp (SEQ ID NO.2). The PCR amplification program was as follows: 95℃ pre-denaturation for 2 min, 95℃ denaturation for 30 s, 57℃ annealing for 10 s, 72℃ extension for 30 s, for 35 cycles, followed by a final extension at 72℃ for 10 min. The PCR products were separated by electrophoresis on a 1% agarose gel, stained with ethidium bromide (EB), photographed, and the results were recorded. The PCR products were then excised and recovered. The electrophoretic bands were recovered using the Agarose Gel DNA PCR Kit (TaKaRa). The PCR products recovered from gel excision were ligated into the pTRV2 vector digested with PstI using the ClonExpress II One Step Cloning Kit (Vazyme) according to the instructions to obtain the pTRV2:SNIPER plasmid. This plasmid was transformed into *E. coli* competent cells JM109, plated on LB agar plates (containing 50 μg / mL kanamycin), and incubated at 37°C for 16 hours. After colony PCR verification, three clones were picked, and the TRV2:SNIPER plasmid was extracted using a plasmid extraction kit (Takara), with the sequence shown in SEQ ID NO.2. The correctly sequenced plasmid was electroporated into *Agrobacterium* GV3101, plated on LB agar plates (Kanamycin 50 μg / mL, Rif 50 μg / mL), and incubated at 30°C for 48 hours. After colony PCR verification, the correct clones were picked for subsequent experiments.

[0052] Example 3. Virus-induced silencing of the tobacco SNIPER gene

[0053] The specific steps are briefly described below:

[0054] 1) Agrobacterium culture

[0055] Single colonies of Agrobacterium GV3101 transfected with the pTRV2:SNIPER vector (SNIPER gene silencing vector), single colonies of Agrobacterium GV3101 containing the pTRV2:GFP vector (control), and single colonies of Agrobacterium GV3101 containing the pTRV1 virus were picked from the plates and inoculated into 2 mL of LB liquid medium (Kan 50 μg / mL, Rif 50 μg / mL) and incubated overnight at 30°C and 200 rpm until the OD600 reached 2.0. The overnight cultured Agrobacterium GV3101 was centrifuged at 3000g for 5 minutes to collect the bacterial cells. The bacterial suspension was resuspended in buffer (components: 10 mM 2-[N-morpholino]ethanes μL fonic acid, 10 mM MgCl2, 200 μM Macetosyringone, pH 5.6) and centrifuged again to collect the bacterial cells. The washing was repeated three times, and the bacterial suspension was diluted with buffer. pTRV1 was mixed 1:1 with pTRV2:SNIPER and pTRV2:GFP Agrobacterium and labeled as TRV:SNIPER and TRV:GFP, respectively, with a final concentration of 1.0 for each.

[0056] 2) Gene silencing in tobacco

[0057] The prepared Agrobacterium tumefaciens bacterial solution was injected into four leaves of two-week-old tobacco seedlings using a syringe. After four weeks of cultivation in a greenhouse (21-23℃, 14h light / 10h dark), the gene silencing level of the treated tobacco seedlings was detected.

[0058] Reference method: Dong, Y., Bμrch-Smith, TM, Liμ, Y., Mamillapalli, P., Dinesh-Kμmar, SP2007. A ligation-independent cloning TRV vector for high-throμghpμtvirμs indμced gene. Plant Physiology, 145, 1161-1170.

[0059] 3) SNIPER silencing efficiency test

[0060] Total RNA was extracted from tobacco leaves after four weeks of silencing. The extraction of total RNA was performed using an Omega RNA extraction kit according to the instructions, and the RNA content and quality were determined using a spectrophotometer.

[0061] First-strand reverse transcription: Using 0.7 μg of RNA as a template, cDNA was synthesized according to the instructions for use of Takara's PrimeScript reverse transcriptase reagent, and the volume was adjusted to 20 μL. The reverse transcription product was diluted 10-fold with water for real-time quantitative PCR to detect gene silencing efficiency.

[0062] Real-time quantitative PCR reaction:

[0063] Pre-quantitative primers:

[0064] 5'-GTTTAGAGGAGTGGGGTGTTG-3'

[0065] Primers after quantification:

[0066] 5'-GGCGTAGGAAACCTTTCGATG-3'

[0067] The PCR reaction system contained 5 μL cDNA, 10 μL SYBR Premix Ex Taq II (Tli RNase H 1 μs), 0.4 μL each of the pre- and post-primer primers, 0.4 μL ROX Reference Dye II, and 13.8 μL water. The reaction program was: Step I: 95°C for 30 seconds, Step II: 95°C for 5 seconds, 60°C for 34 seconds, with 40 cycles in Step II. The melting curve analysis program was: 95°C for 15 seconds, 60°C for 1 minute, 95°C for 15 seconds. Data analysis was performed using 2... -ΔΔCT Methods and results showed that the gene expression level of SNIPER was significantly decreased in tobacco plants with silenced SNIPER compared to those treated with TRV:GFP. Figure 1 However, the plant growth morphology was not significantly altered. Figure 2 ). Reference: Liu, F., Xu, Y., Wang, Y., and Wang, Y. (2018). Real-time PCR Analysis of PAMP-induced marker gene expression in Nicotiana benthamiana. Bio Protoc 8: e3031.

[0068] Example 5. Inoculation of silent tobacco with Phytophthora indica

[0069] Tobacco plants treated with the silencing vector for 4 weeks were placed in a plastic incubator. A drop of 10 μL of sterile water was placed in the center of a flattened leaf, and a fresh *Phytophthora tobaccos* P26 mycelial cake with a diameter of 5 mm was inoculated. The plastic incubator was then sealed to maintain humidity. The inoculated plants were first cultured in the dark for 24 hours, then placed in a greenhouse (21-23℃, 14h light / 10h dark). Three days after inoculation, photographs were taken and leaf disease development was recorded. Compared with the control TRV:GFP-treated plants, the area of ​​lesions on leaves inoculated with *Phytophthora tobaccos* after silencing the SNIPER was significantly reduced. Figure 3 This indicates that the silenced gene SNIPER can significantly improve tobacco resistance to Phytophthora tobaccoii.

[0070] Example 6. Expression of the SNIPER gene in tobacco

[0071] 1) Agrobacterium culture

[0072] Single colonies of Agrobacterium GV3101 transfected with pBin:SNIPER-HA vector and control pBin:eGFP-HA vector, respectively, were picked from the plates in Example 1 and inoculated into 2 mL of LB liquid medium (Kan 50 μg / mL, Rif 50 μg / mL) and incubated overnight at 30°C and 200 rpm on a shaker until the OD600 reached 2.0. The overnight cultured Agrobacterium GV3101 was centrifuged at 3000 g for 5 minutes to collect the cells. The bacterial suspension was resuspended in buffer (components: 10 mM 2-[N-morpholino]ethanesulfonic acid, 10 mM MgCl2, 200 μM Macetosyringone, pH 5.6) and centrifuged again to collect the cells. After washing three times, the bacterial suspension was diluted with buffer. Agrobacterium P19 and pBin::SNIPER-HA or pBin::eGFP-HA were mixed 1:1 to a final concentration of 0.6 for each.

[0073] 2) Tobacco expression of SNIPER-HA

[0074] The prepared Agrobacterium was injected into tobacco leaves using a syringe. After injection, the tobacco was cultured in a greenhouse (21-23℃, 14h light / 10h darkness).

[0075] 3) Detection of cumulative SNIPER-HA protein levels

[0076] Two days after injection, tobacco leaves were collected for protein accumulation detection. The collected tobacco leaves were flash-frozen in liquid nitrogen, ground, and added to protein extraction buffer (components: 150 mM NaCl, 50 mM Tris-HCl pH 7.5, 10 mM ethylenediaminetetraacetic acid, 1.0% (v / v) NP-40, 1 mM phenylmethylsulfonylfluoride, and 1.0% (v / v) protease inhibitor cocktail). The mixture was incubated on ice for 30 minutes. The supernatant (80 μL) was collected by centrifugation at 18000 g and mixed with 20 μL of 5-fold protein loading buffer. The mixture was then boiled in a water bath for 10 minutes. 20 μL of the sample was then electrophoretically separated on an SDS-PAGE gel at 120 V for 1.5 hours. After the reaction, the protein sample was transferred to a PVDF membrane, incubated with 5% PBST milk, and then sealed. After incubating with 1:5000 diluted GFP primary antibody (Abmart) for 2 hours, wash the membrane three times for 5 minutes each time with PBST. Then, add 1:10000 diluted mouse antibody (LI-COR, irdye 800, 926-32210), incubate for 30 minutes, wash the membrane three times for 5 minutes each time with PBST, and scan the membrane for photographs. Figure 4 ).

[0077] 4) Overexpression of SNIPER-HA significantly weakens tobacco resistance to Phytophthora.

[0078] Two days after injection, the leaves were inoculated with Phytophthora indica. Symptoms were observed three days after inoculation. Figure 5 The results were recorded by photograph. Compared with the negative control, tobacco plants overexpressing SNIPER showed significantly larger lesions after inoculation with Phytophthora, confirming that SNIPER overexpression significantly reduced the resistance of tobacco to different Phytophthora fungi.

[0079] Example 7. Inoculation of *Pseudomonas syringae* mutant strain Pto DC3000ΔhopQ1-1

[0080] The pathogenic bacterium used in this study was the *Pseudomonas syringae* mutant strain Pto DC3000ΔhopQ1-1, a variant of wild-type Pto DC3000 lacking the type III effector HopQ1-1, capable of infecting *Nicotiana benthamiana*. The Pto DC3000ΔhopQ1-1 strain was inoculated into LM medium (tryptone 10g, yeast extract 6g, KH2PO4 1.5g, MgSO4 0.35g, NaCl 0.6g, agar 15g l / L, pH 7.0) containing antibiotics (kanamycin 50μg / ml, rifampin 50μg / ml). After incubation at 30℃ for 2 days, the bacterial cells were collected and resuspended in deionized water, adjusting the final OD600 to 0.0002. The prepared bacterial suspension was slowly injected into *Nicotiana benthamiana* leaves from the underside of the leaves using a 1ml syringe without the needle, with sterile water as a control. After 3 days of normal culture, two leaf discs were punched at the injection site using a 0.5 cm diameter punch and placed into 1.5 ml EP tubes. Small steel balls were added, and the mixture was ground at 50 Hz for 30 seconds. Then, 100 μl of sterile water was added, and grinding continued for another 30 seconds. The ground liquid was then diluted according to a specific dilution gradient and spread onto LM medium. The mixture was incubated at 30°C for 2 days, and the number of bacteria per unit area was counted. Compared with the control TRV:GFP-treated plants, the number of bacteria produced by inoculating Pto DC3000ΔhopQ1-1 strain on the leaves of silenced SNIPER was significantly reduced. Figure 6 This indicates that the silenced gene SNIPER can significantly enhance the resistance of tobacco to bacteria.

Claims

1. A gene as represented by SEQ ID NO. 1 SNIPER application in increasing the resistance of tobacco to Phytophthora nicotianae, said application being the silencing or interfering with the expression of the gene SNIPER .

2. The gene shown in SEQ ID NO.1 SNIPER Application in improving tobacco resistance to *Pseudomonas syringae*, wherein the application is silencing or interfering with genes. SNIPER The expression.

3. Silencing the gene shown in SEQ ID NO.1 SNIPER The application of silent fragments in improving tobacco resistance to Phytophthora tobaccois, wherein the application is to silence or interfere with genes. SNIPER The expression.

4. Silencing the gene shown in SEQ ID NO.1 SNIPER The application of silencing fragments in improving tobacco resistance to *Pseudomonas syringae*, wherein the application is to silence or interfere with genes. SNIPER The expression.

5. Contains the gene shown in SEQ ID NO.1 that is silenced. SNIPER The application of gene silencing vectors for silencing fragments in improving tobacco resistance to Phytophthora tobaccois, wherein the application is to silence or interfere with genes. SNIPER The expression.

6. Contains the gene shown in SEQ ID NO.1 that is silenced. SNIPER The application of gene silencing vectors for silencing fragments in improving tobacco resistance to *Pseudomonas syringae*, wherein the application is to silence or interfere with genes. SNIPER The expression.

7. A method for cultivating tobacco varieties resistant to *Phytophthora nicotineae* or *Pseudomonas syringae*, characterized in that, The method is to silence or interfere with genes. SNIPER The expression of the gene SNIPER As shown in SEQ ID NO.1.

Images