PsCAP1, a plant immune-inducing protein, and its applications

Abstract

This invention discloses a plant immune-inducing protein, PsCAP1, and its applications. This protein can induce plant resistance and can be used as a plant immune activator in the control of plant diseases. The amino acid sequence of the protein is shown in SEQ ID NO.2. High concentrations of the protein can be obtained by expression using engineered strains. Treatment of plants with this protein can improve plant resistance to Phytophthora infestans, with low required concentrations, rapid onset of action, and long duration of action. PsCAP1 provides a new approach to improving plant disease resistance and controlling plant diseases.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a plant immune-inducing protein PsCAP1 and its applications. Background Technology

[0002] Phytophthora is a plant pathogenic oomycete that resembles fungi in morphology but differs significantly from fungi in evolutionary relationships and pathogenic mechanisms. It can infect most dicotyledonous plants, and the plant diseases it causes seriously threaten global ecology and food security. Due to the large and complex genome of Phytophthora and the rapid virulence variation in field populations, chemical control remains one of the main methods for controlling plant diseases (Cooke et al., 2012). However, the massive use of chemical fungicides and unscientific application methods have led to a series of problems, including excessive pesticide residues, environmental pollution, and crop damage, seriously affecting sustainable agricultural development and human health.

[0003] In the long-term use of chemical fungicides to control plant diseases, the plant's own immune system has often been overlooked. In recent years, with the development of science, technological progress, and the demands of sustainable development, the application of plant's own immune mechanisms to control plant diseases and pests has increasingly become an important part of plant protection. During the activation of the plant's immune response, pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) is mainly induced by pattern recognition receptors (PRRs) on the cell membrane sensing pathogen-associated molecular patterns (PAMPs) from pathogens or damage-associated molecular patterns (DAMPs) released during host infection, thereby inducing the plant's immune response. (Cao et al., 2014; Jones and Dangl, 2006). These elicitors that can activate plant immunity, being essentially carbohydrates or proteins, are increasingly attracting attention in the field of fungal, bacterial, and viral disease control and application because they can induce plant resistance while reducing environmental pollution (Cao et al., 2014; Petutschnig et al., 2014).

[0004] Developing effective plant immune-inducing proteins can provide effective protein resources for developing novel protein-based biopesticides that activate plant immunity.

[0005] References

[0006] H., Albert, I., Fan, L., Reinhard, A., and Nürnberger, T. (2014). Immune receptor complexes at the plant cell surface. Current opinion in plant biology 20, 47 - 54.

[0007] Cao, Y., Liang, Y., Tanaka, K., Nguyen, C. T., Jedrzejczak, R. P., Joachimiak, A., and Stacey, G. (2014). The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin - induced complex with related kinase CERK1. eLife 3.

[0008] Cooke, D. E., Cano, L. M., Raffaele, S., Bain, R. A., Cooke, L. R., Etherington, G. J., Deahl, K. L., Farrer, R. A., Gilroy, E. M., Goss, E. M., et al. (2012). Genome analyses of an aggressive and invasive lineage of the Irish potato famine pathogen. PLoS pathogens 8, e1002940.

[0009] Jones,JD,and Dangl,JL(2006).The plant immune system.Nature 444,323-329.Petutschnig,EK,Stolze,M.,Lipka,U.,Kopischke,M.,Horlacher,J.,Valerius,O.,Rozhon,W.,Gust,AA,Kemmerling,B.,Poppenberger,B.,et al.(2014).Anovel Arabidopsis CHITIN ELICITOR RECEPTOR KINASE 1(CERK1)mutant with enhanced pathogen-induced cell death and altered receptor processing.Newphytologist 204,955-967. Summary of the Invention

[0010] One of the objectives of this invention is to provide a plant immune-inducing protein, PsCAP1.

[0011] The second objective of this invention is to provide a gene sequence encoding the aforementioned plant immune-inducing protein PsCAP1.

[0012] The third objective of this invention is to provide a recombinant expression vector containing the gene encoding the plant immune-inducing protein PsCAP1.

[0013] The fourth objective of this invention is to provide the application of the above-mentioned plant immune induced protein PsCAP1 and its encoding gene.

[0014] The specific content of this invention is as follows:

[0015] This invention provides a plant immune-inducing protein PsCAP1, the amino acid sequence of which is shown in SEQ ID NO.2, or a protein derived from SEQ ID NO.2 by substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence of SEQ ID NO.2, which is associated with Phytophthora blight.

[0016] This invention also provides the encoding gene for the aforementioned plant immune-inducing protein PsCAP1. Using the amino acid sequence of PsCAP1 from this invention, codon-optimized nucleic acid sequences that are beneficial for expression in *E. coli* can be designed and synthesized.

[0017] As a preferred technical solution, the coding gene is as follows (1) or (2):

[0018] (1) The nucleotide sequence as shown in SEQ ID NO.1;

[0019] (2) A nucleotide sequence having at least 70% homology with SEQ ID NO.1; preferably a nucleotide sequence having at least 80% homology with SEQ ID NO.1; more preferably a nucleotide sequence having at least 85% homology with SEQ ID NO.1; even more preferably a nucleotide sequence having at least 90% homology with SEQ ID NO.1; most preferably a nucleotide sequence having at least 95% homology with SEQ ID NO.1.

[0020] This invention also provides a recombinant vector, expression cassette, or recombinant bacteria containing the above-mentioned coding gene. Preferably, the recombinant vector is a prokaryotic expression vector obtained by inserting the PsCAP1 coding gene into pGEX-4T-2. This recombinant vector can be used to express a fusion protein (GST-PsCAP1) with a molecular weight of approximately 53 kDa in Escherichia coli Rossetta (DE3), which can induce resistance responses in plants such as tobacco, pepper, and tomato, improve plant immunity, and reduce plant damage caused by Phytophthora infestans, Phytophthora capsici, and Phytophthora virosa.

[0021] The present invention also provides the application of the above-mentioned plant immune induction protein PsCAP1 in inducing plant defense responses or improving plant disease resistance.

[0022] The plant immune-inducing protein PsCAP1 described in this invention can effectively induce the expression of disease resistance-related genes NbCYP71D20, NbWRKY7, NbWRKY8, and NbAcre31. Theoretically, for plants expressing these disease resistance-related genes, or for diseases related to these genes, the protein PsCAP1 described in this invention can induce defense or enhance plant disease resistance. In some specific examples, the plants are tobacco, pepper, and tomato. In more specific examples, the disease resistance refers to resistance to Phytophthora tobaccosifolia, Phytophthora capsici, and Phytophthora virosa.

[0023] This invention also provides a method for developing plant disease resistance, which involves spraying the plant immune-inducing protein PsCAP1 described in this invention onto the plant surface or injecting it into the plant leaves. The concentration of the sprayed PsCAP1 is 0.8–1.5 μM. In some specific examples, the plants are tobacco, pepper, and tomato. More preferably, the disease resistance is resistance to Phytophthora tobaccoii, Phytophthora capsici, and Phytophthora virosa.

[0024] The beneficial effects of this invention are as follows:

[0025] This plant immune-inducing protein can significantly improve plant disease resistance, requiring low concentrations, showing rapid effects, and having a long duration of action. PsCAP1 activates the plant's own immune system, providing a new approach to enhancing plant resistance, and therefore has broad application prospects in agricultural production. Attached Figure Description

[0026] Figure 1 Escherichia coli Rossetta (DE3) expressed PsCAP1 protein, and the antibody used for Western blot detection was anti-GST;

[0027] Figure 2 To detect reactive oxygen species (ROS) bursts induced by the plant immune-inducing protein PsCAP1 in tobacco, pepper, and tomato.

[0028] Figure 3 To detect the expression of tobacco disease resistance-related genes induced by the plant immune-inducing protein PsCAP1;

[0029] Figure 4 Plant immune-inducing protein PsCAP1 induces resistance in tobacco to Phytophthora nicotineis. (A) Infection symptoms of PsCAP1-treated and untreated tobacco; (B) Biomass detection of PsCAP1-treated and untreated tobacco after inoculation with Pytophthora nicotineis. Detailed Implementation

[0030] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods 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 Sangon Biotech Co., Ltd.

[0031] Unless otherwise specified, the *Phytophthora sojae* strain used in the following examples is *Phytophthora sojae* strain P6497, which is currently stored in this laboratory, and the inventors and applicant promise to make it permanently available to the public.

[0032] Example 1: Isolation and Identification of PsCAP1, a Plant Immune Inducing Protein

[0033] A suspension of *Phytophthora spp.* zoospores was prepared, and the inoculation concentration was adjusted to 100 spores / μL. Soybean leaves were treated with 0.1% Tween-20, followed by treatment with sterile distilled water to thoroughly remove the Tween-20. Soybean leaves were then immersed in the zoospore suspension for 30 minutes, after which they were gently removed and placed in petri dishes lined with moistened filter paper for 6 hours at 25°C in the dark. The intercellular fluid from the infected soybean leaves was collected using a vacuum pump, and the fraction was further separated and concentrated according to molecular weight using ultrafiltration. The 10kD-30kD fraction, which induced severe cell death, was sent to BGI Genomics in Shenzhen for proteomics analysis. Based on the proteomics data and comparison with the *Phytophthora spp.* protein database, the secreted protein PsCAP1 was identified, and its amino acid sequence is shown in SEQ ID NO.2.

[0034] Example 2: Cloning of the gene encoding the plant immune-inducing protein PsCAP1

[0035] (1) Total RNA extraction:

[0036] Using liquid-cultured Phytophthora soybean mycelia as material, total RNA was extracted from Phytophthora soybean using the Omega RNA Extraction Kit according to the instructions, and the RNA content and quality were detected by spectrophotometer.

[0037] (2) Reverse transcription to generate the first strand:

[0038] Use 0.7 μg of RNA as a template to synthesize cDNA according to the instructions for use of Takara's PrimeScript reverse transcriptase reagent, and bring the volume to 20 μL. Use an appropriate amount of the reverse transcription product for subsequent gene cloning PCR.

[0039] (3) Using the first strand of cDNA as a template for RT-PCR, PCR was performed using standard methods to amplify the full length of the PsCAP1 encoding gene:

[0040] PCR primer amplification sequences:

[0041] Upstream primer:

[0042] 5'-CAGCTAGCATCGATTCCCGGGATGCCTCGCCTCAGTATTTTCG-3'

[0043] Downstream primer:

[0044] 5'-AATCTCTAGAGGATCCCCGGGGAGACTGCCGAAGTCCTGCG-3',

[0045] The 50 μL reaction mixture consisted of 10 μL of 5× buffer, 4 μL of 2.5 mM dNTPs, 0.5 μL of Takara PrimerSTARTaq enzyme, 1 μL of template cDNA, and water to a final volume of 50 μL. The PCR amplification program was: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 1 min, repeated 35 times, followed by a final extension at 72℃ for 10 min. Separation was performed by electrophoresis on an agarose gel, followed by ethidium bromide (EB) staining and photographing. The results were recorded, and the PCR product encoding the PsCAP1 gene was recovered by gel excision. The electrophoretic bands were recovered using the Agarose Gel DNA Purification Kit (TaKaRa). The PCR product of the PsCAP1 encoding gene recovered from gel excision was ligated into the SmaI-digested pGR107::3HA vector (purchased from BioVector Plasmid Vector Culture Collection Center) according to the instructions of the CloneExpress II One Step Cloning Kit (Vazyme) to obtain the pGR107::PsCAP1-3HA plasmid. The plasmid was transformed into E. coli competent cells JM109 and cultured in the dark at 37°C for 16 h on LB agar plates containing 50 μg / mL of Kanamycin. After colony PCR verification, three positive clones were picked, and the plasmid was extracted according to the plasmid extraction kit (Takara). The plasmid was sent to Sangon Biotech Co., Ltd. for sequencing. The sequence obtained should be consistent with the sequence of SEQ ID NO.1.

[0046] Example 3: Prokaryotic expression and purification of plant immune-inducing protein PsCAP1

[0047] (1) Construction of prokaryotic expression vector

[0048] Design specific primers to amplify the PsCAP1 fragment and construct it into the prokaryotic expression vector PGEX-4T-2, where the upstream primer is:

[0049] 5'-GGATCCCCAGGAATTCCCGGGTTCCAGCTCGGAAGTGGCG-3'

[0050] Downstream primer:

[0051] 5'-GGCCGCTCGAGTCGACCGGGGAGACTGCCGAAGTCCTGCG-3'

[0052] (2) Prokaryotic expression and purification of PsCAP1

[0053] The constructed pGEX-4T-2 recombinant vector was transformed into *E. coli* strain Rosetta; single colonies containing the recombinant plasmid were picked and cultured overnight at 37°C and 220 rpm in 2 mL of liquid LB medium containing 50 μg / mL Ampicillin; the overnight cultured *E. coli* culture was then inoculated into 2 mL of liquid LB medium containing 50 μg / mL Ampicillin at a concentration of 1:50–1:100. Ampicillin was cultured in liquid LB medium at 37°C and 220 rpm until OD600 reached 0.6-0.8. 0.5 mM MIPTG (isopropyl-β-D-thiopyranoside) was added to the cultured *E. coli* culture, and the culture was incubated at 16°C and 220 rpm for 8-12 hours to induce protein expression. The induced *E. coli* culture was centrifuged at 5000 rpm for 10 min at room temperature, the supernatant was discarded, and the precipitate was washed three times with sterile 1×PBS at the same speed and centrifugation time. An appropriate amount of sterile 1×PBS was added to the bacterial precipitate to gently resuspend it, and 100 mM PMSF was added and mixed. The *E. coli* suspension was homogenized in a high-pressure homogenizer at low temperature until the solution was clear. The suspension was centrifuged at 8000 rpm for 10 min at room temperature, and an appropriate amount of supernatant was gently aspirated. SDS-PAGE gel electrophoresis was used to examine protein expression and solubility, and subsequent protein purification was performed. The protein was purified using the AKTA™ avant automated protein purifier. 25 (GE Healthcare) purified the target protein in the supernatant. First, the target protein was purified using a GST purification column. The purified collection was then desalted using a desalting column. Subsequently, the protein concentration was measured, and SDS-PAGE electrophoresis, Coomassie brilliant blue staining, and Western blot were performed to detect the purified target protein.

[0054] Results: SDS-PAGE analysis revealed a recombinant PsCAP1 protein (GST-PsCAP1) with an N-terminus containing a GST tag and a molecular weight of approximately 53 kDa. Figure 1 .

[0055] Example 4: Inducing plant immune responses in tobacco, pepper and tomato using the plant immune-inducing protein PsCAP1.

[0056] (1) Prokaryotic expression of plant immune-inducing protein PsCAP1 induces reactive oxygen species bursts in tobacco, pepper and tomato.

[0057] Collect leaf discs from leaves of 5-week-old tobacco, pepper, or tomato plants. The reactive oxygen species background was then removed by floating the plates overnight in 200 μL of sterile water in 96-well plates. A 200 μL reaction buffer containing luminol and peroxidase (35.4 μg / mL luminol, 10 μg / mL peroxidase) and 1 μM purified protein (GST-PsCAP1 or GST) was prepared and replaced with sterile water under light-protected conditions. Luminescence was measured using a GLOMAX96 microplate spectrophotometer (Promega, Madison, WI, USA).

[0058] Results: 1 μM PsCAP1 protein can induce reactive oxygen species (ROS) bursts in tobacco, pepper, and tomato. Figure 2 ).

[0059] (2) Prokaryotic expression of plant immune-inducing protein PsCAP1 significantly increased the transcriptional level of tobacco disease resistance-related genes.

[0060] Five-week-old tobacco leaves from the middle section were selected, and 1 μM PsCAP1 protein was injected into different leaves from the abaxial surface. GST protein of the same concentration was used as a control. Samples were collected 6 hours after injection for analysis of the transcriptional levels of resistance-related genes. Total RNA was extracted using an Omega RNA extraction kit according to the instructions, and its 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 transcription levels.

[0062] The primers used in the real-time quantitative PCR reaction are as follows:

[0063] NbCYP71D20 upstream primer:

[0064] 5'-GTTGACGCCATTGTTGAG-3'

[0065] NbCYP71D20 downstream primer:

[0066] 5'-ATCTTCGCCTCCTAATGC-3'

[0067] NbAcre31 upstream primer:

[0068] 5'-AATTCGGCCATCGTGATTCTTGGTC-3'

[0069] NbAcre31 downstream primer:

[0070] 5'-GAGAAACTGGGATTGCCTGAAGGA-3'

[0071] NbWRKY7 upstream primer:

[0072] 5'-CACAAGGGTACAAACAACACAG-3'

[0073] NbWRKY7 downstream primer:

[0074] 5'-GGTTGCATTTGGTTCATGTAAG-3'

[0075] NbWRKY8 upstream primer:

[0076] 5'-AACAATGGTGCCAATAATGC-3'

[0077] NbWRKY8 downstream primer:

[0078] 5'-TGCATATCCTGAGAAACCATT-3'

[0079] NbEF1α upstream primer:

[0080] 5'-GTATGCCTGGGTGCTTGAC-3'

[0081] NbEF1α downstream primer:

[0082] 5'-ACAGGGACAGTTCCAATACCA-3'

[0083] The PCR reaction system contained 5 μL cDNA, 10 μL SYBR Premix Ex Taq II (Tli RNase H Plus), 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℃, 30 s; Step II: 95℃, 5 s, 60℃, 34 s; Step II was repeated 40 times. The melting curve analysis program was: 95℃, 15 s, 60℃, 1 min, 95℃, 15 s. Data analysis was performed using 2... -ΔΔCT method.

[0084] Results: Quantitative real-time PCR results showed that treatment of tobacco leaves with 1 μM PsCAP1 protein for 6 h significantly induced an increase in the expression of tobacco disease resistance-related genes. Figure 3 ).

[0085] Example 5: Plant immune-inducing protein PsCAP1 enhances the disease resistance of tobacco.

[0086] Using a 1 mL syringe without a needle, 1 μM PsCAP1 protein was injected into different leaves from the abaxial surface, with GST protein of the same concentration used as a control. Leaf samples were inoculated with *Phytophthora nicotineae* 24 h after protein treatment. Infection was observed and samples were collected 3 days after inoculation for genomic analysis. *Phytophthora nicotineae* biomass was detected using quantitative real-time PCR (method as in Example 4). PAR was selected as the *Phytophthora nicotineae* gene, and EF1α as the tobacco gene. The primers used for the quantitative real-time PCR reaction are as follows:

[0087] PAR upstream primer:

[0088] 5'-ATGAACTTCCGCGCTCTGTT-3'

[0089] PAR downstream primer:

[0090] 5'-CAGTGACGCGCACGTAGAC-3'

[0091] NbEF1α upstream primer:

[0092] 5'-GTATGCCTGGGTGCTTGAC-3'

[0093] NbEF1α downstream primer:

[0094] 5'-ACAGGGACAGTTCCAATACCA-3'

[0095] Results: Compared with the negative control GST protein treatment, the number of Phytophthora nephroticis lesions on tobacco leaves treated with PsCAP1 was significantly reduced. Figure 4 A) The biomass of *Phytophthora infestans* infection was significantly reduced. Figure 4 B).

Claims

1. The application of the plant immune-inducing protein PsCAP1 shown in SEQ ID NO.2 in improving the resistance of plants to Phytophthora indica, wherein the plant is tobacco, pepper or tomato.

2. A method for improving plant resistance to Phytophthora indica, characterized in that, The method involves spraying the plant immune-inducing protein PsCAP1, as shown in SEQ ID NO.2, onto the surface of a plant or injecting it into the leaves of a plant, such as tobacco, pepper, or tomato.

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