Fungal elicitor stom and its use in inducing plant immunity and disease resistance

Abstract

The present application relates to the field of plant protection and biological control, and particularly relates to a fungal elicitor STOM and its application in inducing plant immunity and disease resistance. The present application provides a pyricularia oryzae elicitor protein STOM and its coding gene, wherein the elicitor protein is an amino acid as shown in SEQ ID NO: 2, and the coding gene STOM is a nucleotide molecule as shown in SEQ ID NO: 1; and the present application also provides the pyricularia oryzae elicitor STOM and its application in inducing plant immunity and disease resistance. The present application proves through experiments that the elicitor protein can significantly activate plant defense response, such as active oxygen burst, defense gene expression and MAPK activation; and can improve the disease resistance of nicotiana benthamiana to botrytis cinerea, sclerotinia sclerotiorum and other diseases, resist pathogenic bacteria infection, and effectively prevent or reduce the occurrence of diseases. The elicitor protein provided by the present application provides new material resources for the research and development of protein pesticides / plant immunity elicitors, provides materials and theoretical guidance for the compounding of biological pesticides, and has a wide application prospect in agricultural production.

Description

Technical Field

[0001] This invention relates to the fields of plant protection and biological control, and in particular to the fungal elicitor STOM and its application in inducing plant immunity and disease resistance. Background Technology

[0002] Through long-term co-evolution with pathogenic microorganisms, plants have developed a complex and highly efficient immune system. The first line of defense in this immune system is pattern-triggered immunity (PTI), whose core mechanism involves the recognition of pathogen-associated molecular patterns (MAMPs) by pattern recognition receptors on the surface of plant cells. This activates downstream signaling pathways, triggering defensive responses, including reactive oxygen species bursts, MAPK cascade activation, defense gene expression, and programmed cell death. Breeding resistant varieties of certain crops to specific diseases is extremely difficult, and the use of chemical pesticides often leads to environmental pollution and food safety issues. Therefore, the development of protein pesticides (also known as plant immune inducers) based on MAMPs shows promising application prospects.

[0003] Bacterial hairpin proteins have been developed into commercial protein pesticides for field control, with good control effects. MAMPs reported in fungi are mainly chitin and oligosaccharides, components of the fungal cell wall, as well as protein elicitors such as NLP, XEG1, and SGP1. Although the discovery of these MAMPs has advanced our understanding of plant immune mechanisms and provided important resources for the development of immune inducers, existing MAMPs still have the following problems: (1) Limited variety: There are not many fungal MAMPs that can stably induce immune responses, limiting their application scope; (2) Insufficient broad spectrum: Some MAMPs can only function in specific plant genera and species, lacking universality; such as NLP proteins; (3) Limited induction effect: Under field conditions, the immune effect induced by some MAMPs is not strong enough or has a short duration, making it difficult to meet the needs of large-scale crop application; (4) Preparation and stability issues: Some MAMPs have small molecular weights or unstable properties, making it difficult to produce, preserve and use on a large scale; (5) Insufficient commercial application: Currently, most of the existing immune inducers at home and abroad are concentrated on oligosaccharide molecules, and there is a lack of widespread application examples of protein MAMPs.

[0004] Therefore, there is an urgent need to identify new fungal-derived elicitors, especially protein molecules that can effectively induce immunity and significantly enhance plant disease resistance. Summary of the Invention

[0005] To overcome the aforementioned technical problems, this invention provides a novel fungal secretory protein, STOM. This protein possesses a typical TOM20 domain and its conserved C-terminal amino acid sequence, enabling it to... Nicotiana benthamiana The study found that the novel coronavirus (Nicotiana benthamiana) induces a typical immune response and significantly enhances resistance to fungal diseases, overcoming the limitations of existing elicitors in terms of limited types and insufficient function. This provides an effective material source for developing broad-spectrum, highly efficient, and environmentally friendly immune protein biopesticides.

[0006] The first aspect of this invention provides a fungal elicitor protein STOM, wherein the elicitor protein STOM is a protein of the following type 1), 2), 3), or 4): 1) A protein consisting of the amino acid sequence shown in SEQ ID NO:2; 2) Proteins derived from SEQ ID NO:2 with one or more amino acid residues substituted and / or deleted and / or added, and having elicitor protein function.

[0007] 3) The amino acid sequence has more than 90% homology with 1) or 2) and encodes an amino acid sequence that induces plant defense responses and enhances plant disease resistance; 4) Other fungi have more than 70% of the amino acid sequences that are the same as 1) or 2), and the encoded proteins have amino acid sequences that induce plant defense responses and enhance plant disease resistance.

[0008] In some embodiments, the amino acid sequence of the other fungi in 4) having more than 70% of the amino acid sequence of 1) or 2) and encoding a protein that induces plant defense response and enhances plant disease resistance is selected from any of the amino acid sequences shown in SEQ ID NO:13-18.

[0009] A second aspect of the present invention provides a fungal elicitor protein encoding gene STOM, wherein the encoding gene STOM is a DNA molecule as shown in 1) or 2) or 3) or 4) below: 1) The DNA molecule shown in SEQ ID NO:1 of the sequence listing; 2) The nucleotide sequence encoding the protein as described in the first aspect; 3) A DNA molecule that hybridizes to the DNA sequence defined in 1) or 2) under stringent conditions and encodes the fungal elicitor protein STOM; 4) Nucleotide sequences that have more than 90% homology with nucleotide sequences of 1) or 2) and encode proteins that induce plant defense responses and enhance plant disease resistance.

[0010] In some embodiments, the nucleotide sequence of 4) having more than 90% homology with the nucleotide sequence of 1) or 2) and encoding a protein that induces plant defense responses and enhances plant disease resistance is selected from any of the nucleotide sequences shown in SEQ ID NO:19-24.

[0011] A third aspect of the present invention provides a vector containing the STOM encoding gene as described in the second aspect.

[0012] In this invention, the vector enables the target gene to be used for transformation and expression. The preferred expression vector is pHMTc, which is a recombinant vector in which the target gene is inserted.

[0013] A fourth aspect of the present invention provides a host cell containing a vector as described in the third aspect.

[0014] In this invention, the recombinant vector can be expressed using Escherichia coli Rosetta.

[0015] The fifth aspect of the present invention provides the application of the elicitor protein STOM or the encoding gene STOM in stimulating plant defense responses, hypersensitive responses and / or stimulating the expression of plant disease resistance genes.

[0016] In this invention, the disease resistance refers to the ability to resist diseases caused by Sclerotinia sclerotiorum and Botrytis cinerea.

[0017] The sixth aspect of the present invention provides the application of the elicitor protein STOM or the encoding gene STOM in improving plant resistance to diseases.

[0018] In some embodiments, the disease is caused by Sclerotinia sclerotiorum and Botrytis cinerea.

[0019] The seventh aspect of the present invention provides a method for improving plant disease resistance, characterized in that the method includes spraying or injecting the elicitor protein STOM described in the first aspect or the encoding gene STOM described in the second aspect onto plant tissues.

[0020] In some embodiments, the disease resistance is the ability to resist diseases caused by Sclerotinia sclerotiorum and Botrytis cinerea.

[0021] Compared to existing technologies, this invention is the first to identify the elicitor STOM from a secreted protein of *Strombus oryzae*, construct it into the plant expression vector PVX, transform it with *Agrobacterium*, and inject it into tobacco. It was found that STOM induced cell death in *Nicotiana benthamiana*. Furthermore, this protein was discovered to be fungus-specific, widely present in fungi, and capable of inducing cell death in most fungi. Through protein treatment and *Agrobacterium*-mediated transient transformation, it was found that in addition to inducing cell death, STOM also induced reactive oxygen species bursts, defense gene expression, and MAPK activation in *Nicotiana benthamiana*, and enhanced the resistance of *Nicotiana benthamiana* to *Sclerotinia sclerotiorum* and *Botrytis cinerea*. Therefore, the elicitor protein STOM can be used as a novel immune inducer for plant disease control, possessing advantages such as safety, environmental friendliness, and broad-spectrum disease resistance, and has significant application prospects. Attached Figure Description

[0022] Figure 1 This is a gel electrophoresis image of the protein after STOM purification.

[0023] Figure 2 This figure shows the cell death induced by STOM and its fungal homologous proteins.

[0024] Figure 3 The figure shows the results of STOM-induced reactive oxygen species production in Benzoic acid.

[0025] Figure 4 The image shows the results of STOM-induced expression of the defense gene in Nicotiana benthamiana.

[0026] Figure 5 The image shows the results of STOM-induced activation of MAPK in Smoke Benedict's tobacco.

[0027] Figure 6 The image shows the results of STOM-induced resistance of *Nyctaginus benthamiana* to *Sclerotinia sclerotiorum*.

[0028] Figure 7 The figure shows the results of STOM-induced resistance of *Fumica benthamiana* to *Botrytis cinerea*.

[0029] Figure 8 This figure shows the cell death induced by STOM homologs in other fungi. Detailed Implementation

[0030] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention is further described below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available. Example 1

[0031] The protein elicitor STOM was used, the nucleotide sequence of which is shown in SEQ ID NO:1 in the sequence listing, and the amino acid sequence of which is shown in SEQ ID NO:2.

[0032] (1) Construction of prokaryotic expression vectors and plant expression vectors Specific primers encoding the STOM elicitor gene of *Aspergillus oryzae* were designed: forward primer: 5'-ctgtattttcagggcgaattcCTCGTCGCTTATGCCCTGTAC-3' (SEQ ID NO:3), reverse primer: 5'-caggtcgactctagaggatccTCAGTCAAGACCGACGTTGGG-3' (SEQ ID NO:4). Using *Aspergillus oryzae* cDNA as a template, the full-length STOM gene was amplified (94℃ 5min; 94℃ 30s, 58℃ 30s, 72℃ 30s, 34 cycles; 72℃ 5min). The pHMTc vector was digested with EcoRI and BamHI, and the STOM fragment was cloned into the pHMTc vector using recombinant cloning. The recombinant vector was transformed into *Escherichia coli* DH5α strain, plated on agar plates containing ampicillin, and positive clones were screened by colony PCR. Plasmids were extracted from the screened positive clones by shaking to obtain the prokaryotic expression recombinant vector.

[0033] Specific primers encoding the STOM elicitor gene of *Aspergillus oryzae* were designed: forward primer: 5'- gaacgatagggtacccccgggATGGTTCAGACGTCAACTGTGGT-3' (SEQ ID NO:5), reverse primer: 5'- gcccttgctcaccatggatccGTCAAGACCGACGTTGGGG-3' (SEQ ID NO:6). Using *Aspergillus oryzae* cDNA as a template, the full-length STOM gene was amplified (94℃ 5 min; 94℃ 30 s, 58℃ 30 s, 72℃ 30 s, 34 cycles; 72℃ 5 min). The pBINHA vector was digested with Sma I and BamHI, and the STOM fragment was cloned into the pBINHA vector using recombinant cloning. The recombinant vector was transformed into *Escherichia coli* DH5α strain, plated on agar plates containing kanamycin, and positive clones were screened by colony PCR. Plasmids were extracted from the screened positive clones by shaking to obtain the plant expression recombinant vector.

[0034] Results: The prokaryotic expression vector of STOM was obtained through colony PCR and sequencing verification.

[0035] (2) Inducing protein expression The recombinant plasmid was introduced into *E. coli* Rosetta expression host and plated on selective medium containing ampicillin and chloramphenicol. Positive clones were screened by colony PCR. Positive strains were picked and inoculated into 4 mL of LB medium containing both antibiotics, incubated overnight at 37°C with shaking, and then transferred to 200 mL of LB liquid medium with the same antibiotic conditions. The culture was continued until the OD600 reached 0.6–0.8. Subsequently, 200 μL of 0.1 M IPTG was added, and the culture was induced overnight at 28°C with shaking.

[0036] (3) Purification of recombinant proteins The induced bacterial cells were collected in 50 mL centrifuge tubes and centrifuged at 8000 rpm for 10 min at 4 °C. The cells were resuspended in 10–20 mL of lysis buffer (NaH₂PO₄ 50 mM, NaCl 300 mM, Imidazole 10 mM, pH 8.0) and 20 μL of lysozyme was added. The mixture was incubated on ice for 30 min. The cells were then sonicated (1 s / 3 s intervals, 30 min), followed by centrifugation at 10000 rpm for 70 min at 4 °C. The supernatant was collected. A 400 μL nickel column was washed twice with 10 mL of lysis buffer, and the supernatant was added. The column was incubated on a shaker at 4 °C for 2 h. After incubation, the supernatant was removed by centrifugation at 3210 rpm for 5 min. The sample was then washed three times sequentially with 20 mM elution buffer (NaH₂PO₄ 50 mM, NaCl 300 mM, Imidazole 20 mM, pH 8.0), followed by three washes with 50 mM elution buffer (Imidazole 50 mM), and finally eluted 10 times consecutively with 250 μL elution buffer (Imidazole 250 mM). 10 μL of the eluent was added to 6× SDS loading buffer, and the mixture was boiled in a water bath for 10 min before SDS-PAGE analysis. The purification was assessed using Coomassie Brilliant Blue R250 staining. The purified product was then transferred to imidazole-free Tris buffer via ultrafiltration (Millipore Amicon Ultra-15c) for subsequent experiments.

[0037] Results: Purified STOM recombinant protein was successfully obtained. SDS-PAGE analysis showed that the purified fusion protein band was single and consistent with the theoretical molecular weight (see appendix). Figure 1 ).

[0038] Example 2 STOM-induced plant defense response (1) Inducing tobacco allergic reactions STOM was constructed into a plant expression vector and then transformed into Agrobacterium GV3101 strain. After 36 hours of shaking, the OD value was adjusted to 1 OD. Approximately 4-week-old *Nicotiana benthamiana* plants were selected, and the bacterial culture was injected into the leaves from the underside using a 1 ml syringe without a needle. Simultaneously, a GFP-expressing vector was used as a control, and the reported cell death elicitor SGP1 was used as a negative control. Two days after injection, cell death was observed and photographed.

[0039] Approximately 4-week-old Nicotiana benthamiana plants were selected. Using a 1 ml syringe without a needle, 1 μM STOM recombinant protein was injected into the Nicotiana benthamiana leaf from the back. Simultaneously, 1 μM MBP was used as a control. Placental blue staining was performed and photographs were taken 48 hours after injection.

[0040] Results: Both Agrobacterium-mediated transient transformation and direct injection of 1 μM recombinant protein induced cell death in *Nyctaginis benthamiana*; placental blue staining stained the allergic reaction sites blue, such as... Figure 2 As shown in AB.

[0041] (2) Inducing ROS production in tobacco leaves Inject 1 μM STOM protein or Agrobacterium containing a plant expression vector into leaves of Nicotiana benthamiana. Place the treated leaves in DAB staining solution (1 mg / ml, pH=3.8) and treat at room temperature in the dark for 8 hours. Remove the staining solution and add anhydrous ethanol for decolorization. After all the green color of the leaves has been removed, take out the leaves and photograph them.

[0042] The leaves of the tobacco plant were punched into several small discs using a puncher. They were then placed in a 96-well plate containing deionized water and left to stand overnight. The deionized water was then replaced with a reaction solution containing 1 μM STOM recombinant protein (L-012: 10 μg / mL; Horseradish peroxidase 10 μg / mL). The solution was then quickly placed into a microplate and the relative amount of ROS generated was measured.

[0043] Results: After injection of STOM recombinant protein and Agrobacterium containing the STOM-expressing plant expression vector into leaves, obvious brown deposits appeared at the injection site, indicating that STOM recombinant protein induced ROS production in Nicotiana benthamiana leaves. Figure 3 As shown in Figure AB, the L-012 assay revealed that STOM recombinant protein began to induce ROS production approximately 5 minutes after treatment, reaching a peak at about 20 minutes. Figure 3 As shown in C.

[0044] (3) Inducing the expression of resistance-related genes After injecting 1 μM STOM recombinant protein and a mock control into tobacco leaves, samples were taken at different time points. RNA was extracted using a plant RNA extraction kit, and genomic DNA was removed to obtain high-purity RNA. First-strand cDNA was synthesized using a reverse transcription kit. Following the instructions of the quantitative PCR kit, 2 μL of the reverse transcription product was used as a template, and then real-time quantitative PCR was performed. EF-1α As an internal reference gene, it is related to genes such as those for resistance to Nicotiana benthamiana. NbPR1a and NbPR4 The expression level was measured.

[0045] The primers used are as follows: NbEF1a-QF: 5'-AGAGGGCCCTCAGACAAAC-3' (SEQ ID NO: 7); NbEF1a-QR: 5'-TAGGTCCAAAGGTCACAA-3' (SEQ ID NO: 8); NbPR1a-QF: 5'-CCGCCTTCCCTCAACTCAAC-3' (SEQ ID NO: 9); NbPR1a-QR: 5'-GCACAACCAAGACGTACTGAG-3' (SEQ ID NO: 10); NbPR4-QF: 5'-GGCCAAGATTCCTGTGGTAGAT-3' (SEQ ID NO: 11); NbPR4-QR: 5'-CACTGTTGTTTGAGTTCCTGTTCCT-3' (SEQ ID NO: 12).

[0046] Results: Quantitative real-time PCR results showed that STOM protein could be significantly induced after injection of Nicotiana benthamiana. NbPR1a The expression of [the substance] was significantly induced at 12 hpi, 36 hpi, and 48 hpi after treatment. NbPR4 The expression is as follows Figure 4 As shown.

[0047] (4) Inducing MAPK activation The leaves of *Tobacco Benedict* were punched into several small discs and placed in a 96-well plate containing deionized water. After standing overnight, the deionized water was replaced with a reaction solution containing 1 μM STOM recombinant protein. Samples were taken at different time points, and total protein was extracted using lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 1% SDS, 0.5 mM EDTA, 1× PMSF, and 1× protease inhibitor cocktail). The phosphorylation level was then detected by Western blot using an anti-phospho-p44 / 42 MAPK (pTEpY) antibody.

[0048] Results: STOM induced MAPK activation after 5 minutes of processing, reaching its peak after 10 minutes of processing. Figure 5 As shown.

[0049] Example 3: STOM-induced resistance in tobacco to Sclerotinia sclerotiorum and Botrytis cinerea. (1) Inducing tobacco resistance to Sclerotinia sclerotiorum. After STOM was expressed in Nicotiana benthamiana, it was inoculated with Sclerotinia sclerotiorum and cultured in a moist environment. The diameter of the lesions was then counted.

[0050] Results: STOM significantly reduced Sclerotinia sclerotiorum infection, indicating that STOM induced resistance of Nicotiana benthamiana to Sclerotinia sclerotiorum. Figure 6 As shown.

[0051] (2) Inducing tobacco resistance to Botrytis cinerea After STOM was expressed in *Fumica benthamiana*, it was inoculated with *Botrytis cinerea* and cultured in a moist environment. The diameter of the lesions was then counted.

[0052] Results: STOM significantly reduced Botrytis cinerea infection, indicating that STOM induced resistance of *Fumica benthamiana* to *Fumica benthamiana*. Figure 7 As shown.

[0053] Example 4: Other fungal STOM homologs induce Nicotiana benthamiana cell death. By searching other pathogenic fungal protein libraries via BLAST, STOM homologous proteins were found in other fungi. These STOM homologous proteins were expressed in *Nicotiana benthamiana* using an Agrobacterium-mediated transient transformation method, and their induction of *Nicotiana benthamiana* cell death was observed and statistically analyzed periodically.

[0054] The IDs, species, and sequences of homologous proteins from other fungal species mentioned above are shown in the table below.

[0055]

[0056] Results: Except for STOM in *Synthia spp.*, most STOM homologs in other fungi were able to induce cell death in *Nicotiana benthamiana*, such as... Figure 8 As shown.

[0057] The present invention has been described above by way of example. It should be noted that any simple modifications, alterations or other equivalent substitutions that can be made by those skilled in the art without creative effort without departing from the core of the present invention fall within the protection scope of the present invention.

Claims

1. The application of the elicitor protein STOM or its encoding gene STOM, characterized in that, The application is any of the following: 1) Application in stimulating tobacco defense responses, allergic responses, and / or stimulating the expression of tobacco disease resistance genes; 2) Application in improving tobacco's resistance to diseases caused by Sclerotinia sclerotiorum and Botrytis cinerea; The amino acid sequences of the elicitor protein STOM are shown in SEQ ID NO:2 and SEQ ID NO:13-18, respectively. The nucleotide sequences encoding the STOM gene are shown in SEQ ID NO:1 and SEQ ID NO:19-24, respectively. The application includes spraying or injecting the elicitor protein STOM or its encoding gene STOM into tobacco tissue.

2. A method for improving the disease resistance of tobacco, characterized in that, The method includes spraying or injecting the elicitor protein STOM or its encoding gene STOM into tobacco tissue; the disease resistance is the ability to resist diseases caused by Sclerotinia sclerotiorum and Botrytis cinerea, the amino acid sequences of the elicitor protein STOM are shown in SEQ ID NO:2 and SEQ ID NO:13-18, respectively; the nucleotide sequences of the encoding gene STOM are shown in SEQ ID NO:1 and SEQ ID NO:19-24, respectively.

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