Trichoderma asperellum Cerata-platanin protein CP3 and application thereof

Abstract

The invention relates to a trichoderma asperellum sourced Cerata-plaatanin (CP) family protein CP3 and application thereof, and belongs to the technical field of biological prevention and control of plant diseases. The CP3 protein is derived from Trichoderma asperellum, belongs to a Cerato-platanin family and has a conservative CP structural domain, the amino acid sequence registration number of the CP3 protein is XP024756165.1, and the registration number of a coding gene of the CP3 protein is 36612810 (Gene ID). The protein is a secreting type protein, has signal peptide and can be positioned in tomato cell nucleuses. The invention proves that the disease spot area of tomatoes infected with Botrytis cinerea can be reduced by 71% after the CP3 protein is exogenously applied; after tomatoes are treated by the trichoderma asperellum CP3 overexpression strain, the area of gray mold scabs is remarkably reduced, and the CP3 gene knockout strain has no disease-resistant effect. The CP3 protein and the trichoderma asperellum strain expressing the CP3 protein can be used for preparing biological agents for enhancing the botrytis cinerea resistance, application modes comprise rhizosphere application, root system inoculation and the like, adverse effects on tomato growth are avoided, and a new effective means is provided for green prevention and control of the botrytis cinerea.

Description

Technical Field

[0001] This invention relates to a novel use of Cerato-platanin protein in plants, specifically to the application of Cerato-platanin protein CP3 in inducing disease resistance in tomatoes. Background Technology

[0002] Trichoderma ( Trichoderma Trichoderma sp. (spp.) is a class of beneficial fungi widely found in nature. They are important biocontrol fungi in the biological control of plant diseases and play a vital role in sustainable agricultural development. These fungi can establish interactions with plants, secreting various effector proteins and other signaling molecules to stimulate the plant's own immune defense system, thereby enhancing the plant's resistance to pathogens. Studies have shown that effector molecules such as hydrophobic proteins secreted by Trichoderma sp. can induce systemic resistance in plants by affecting signaling pathways such as jasmonic acid (JA) and ethylene (ET). This provides an effective approach for the green control of plant diseases. However, many effector proteins with disease resistance potential have yet to be discovered and applied.

[0003] Tomato gray mold is caused by Botrytis cinerea (… Botrytis cinerea Gray mold is a global fungal disease caused by infection, severely affecting the yield and quality of tomatoes. Currently, the control of tomato gray mold mainly relies on chemical agents, but chemical pesticides easily cause environmental pollution and excessive pesticide residues, which runs counter to the development concept of green agriculture.

[0004] Cerato-platanin (CP) family proteins are a class of small, cysteine-rich secretory proteins unique to fungi, exhibiting a unique "dual activity" in fungal-plant interactions. CP proteins from pathogenic fungi are mostly virulence factors, while CP proteins from beneficial fungi mostly act as immune inducers, stimulating plant defense responses.

[0005] Chemical control of tomato gray mold has many shortcomings, and biological control is an important direction for sustainable prevention and control. As a beneficial fungus, *Trichoderma echinococcus* secretes CP proteins with potential application value in disease resistance; however, the discovery, verification of disease resistance functions, and application research of novel effector proteins in this family are still relatively scarce. Therefore, screening for highly effective CP family proteins in *Trichoderma echinococcus* and clarifying their application value is of great significance for developing novel biological control technologies for tomato gray mold and promoting the development of green agriculture. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing chemical fungicides in the control of tomato gray mold, which easily lead to drug resistance and cause environmental pollution, as well as the lack of discovery and application of highly effective disease-resistant effector proteins in the Trichoderma CP family. This invention provides Trichoderma CP family protein CP3, overexpression strains, and related applications.

[0007] The first aspect of this invention is to provide a Cerato-platanin (CP) family protein CP3 from *Trichoderma acicularis*, wherein CP3 contains a conserved CP domain and is highly conserved evolutionarily within the *Trichoderma* genus; its encoding gene has the NCBI Gene ID: 36612810, and the NCBI accession number for the encoded protein is XP_024756165.1; this protein is a secreted protein with a signal peptide that can be localized to the tomato cell nucleus, and can enhance the tomato's resistance to *Botrytis cinerea* by activating the jasmonic acid / ethylene (JA / ET) signaling pathway and generating reactive oxygen species (ROS). Botrytis cinerea Resistance to ).

[0008] The second aspect of this invention is to provide a recombinant Trichoderma strain that overexpresses CP3. The strain is obtained by inserting the CP3 encoding gene described in the first aspect of this invention into an expression vector containing a strong gpdA promoter, followed by PEG-mediated protoplast transformation into wild-type Trichoderma. Its CP3 expression level is 3-4 times that of the wild type, which can reduce the area of ​​gray mold lesions on tomatoes and has no adverse effects on tomato growth.

[0009] A third aspect of the present invention is to provide a method for constructing the *Trichoderma hygroscopica* strain overexpressing the strain described in the second aspect of the present invention, comprising the following steps: Using Trichoderma genomic DNA as a template, the CP3 coding gene was amplified; the amplified CP3 coding gene was ligated into a vector containing a strong gpdA promoter to construct a recombinant expression vector; the recombinant expression vector was introduced into wild-type Trichoderma protoplasts, and after resistance screening and RT-qPCR verification, recombinant strains with CP3 expression levels 3-4 times higher than those of wild-type strains were obtained.

[0010] The fourth aspect of this invention provides the application of the CP3 protein described in the first aspect of this invention or the overexpression of *Trichoderma echinococcus* strain described in the second aspect of this invention in the control of tomato gray mold: When the CP3 protein is applied at a concentration of 3 μg / mL, the area of ​​tomato gray mold lesions is reduced by ≥71%; when the tomato variety is Moneymaker (MM), the area of ​​lesions in wild-type tomatoes is reduced by 45%; when the tomato variety is Micro-Tom (MT), the area of ​​lesions in tomatoes is reduced by 50%. The overexpression of *Trichoderma echinococcus* strain is applied at a concentration of 1×10... 6When CFU / mL was applied, the area of ​​gray mold lesions in tomatoes was significantly reduced compared to the wild-type strain treatment group, while the lesion area of ​​the CP3 gene knockout strain treatment group was significantly increased, and the disease resistance of the supplemented strain was restored to the level of the wild type.

[0011] The fifth aspect of this invention provides a method for preventing and controlling gray mold in tomatoes, comprising applying the CP3 protein described in the first aspect of this invention or the overexpressing Trichoderma strain described in the second aspect of this invention to tomatoes; the application method is root irrigation (CP3 protein or strain spore suspension), preferably at the four-leaf stage or four-week hydroponic stage of tomatoes. Attached Figure Description

[0012] Figure 1 This is an expression profile of the Trichoderma CP3 gene in the Trichoderma-tomato interaction of the present invention. Figure 2 Phylogenetic tree diagram and protein verification of the CP3 protein of this invention. a: Phylogenetic tree diagram; b: SDS-PAGE electrophoresis and Western blot verification diagram of CP3 protein.

[0013] Figure 3 This diagram illustrates the effect of the exogenous CP3 protein of the present invention on resistance to tomato gray mold. Figure 4 This diagram illustrates the secretory function verification of the CP3 protein of this invention. Figure 5 This is a diagram showing the subimmunogold electron microscopy detection results of the CP3 protein of the present invention. Figure 6 Molecular validation diagram of the CP3 recombinant Trichoderma acicularis strain constructed according to the present invention. a: Electrophoresis diagram of PCR verification of CP3 knockout strain; b: Bar graph of RT-qPCR verification of CP3 expression level in knockout strain; c: Bar graph of RT-qPCR verification of CP3 expression level in overexpression strain; d: Bar graph of RT-qPCR verification of CP3 expression level in complementary strain. Figure 7 This diagram illustrates the effect of the CP3 recombinant Trichoderma acicularis strain of the present invention on resistance to tomato gray mold. a: Comparison of tomato leaf lesion phenotypes after treatment with wild-type, knockout, overexpression, and complementary strains; b: Bar chart of lesion area statistics.

[0014] Figure 8 The diagram shows the experimental verification of the CP3 protein's resistance to tomato gray mold in different tomato varieties. Detailed Implementation

[0015] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0016] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0017] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0018] The main materials and reagents used in this invention are: Strains and plant materials: Trichoderma hygroscopicum ( Trichoderma asperellum DQ-1 strain, used for amplifying the CP3 encoding gene and constructing recombinant strains; Botrytis cinerea (… Botrytis cinerea The strain B05.10 was used to verify the disease resistance of tomatoes. The tomato varieties were Moneymaker (MM) and Micro-Tom (MT). The growing conditions were 25℃, 12h light / 12h dark light cycle, and humidity of 90%±5%.

[0019] Expression vectors: fungal expression vectors containing strong gpdA promoters, prokaryotic expression vectors pGEX6p and pMalc2X, CRISPR-Cas9 knockout vectors (with HYG resistance marker, screening concentration 350 μg / mL), and complementary vectors (with G418 resistance marker, screening concentration 450 μg / mL).

[0020] Main reagents: CTAB extraction kit, SteadyPure Universal RNA Extraction Kit II (AG21022), Evo M-MLV Plus 1st Strand cDNA Synthesis Kit (AG11615), SYBR GreenPremix Pro Taq HS qPCR Kit I (AG11739), etc., were all purchased from Accurate Biotechnology (Hunan) CO.,LTD; G418, Hygromycin (HYG), IPTG, etc. were purchased from commercial channels; Anti-His antibody, Anti-GST antibody, Anti-myc antibody, etc. were purchased from commercial brands such as Proteintech and HUABIO; Yeast signal sequence capture kit (YH7001) was purchased from Coolaber.

[0021] Specific operation method: 1. Identification and Preparation of CP3 Protein: Transcriptome analysis was used to screen key genes involved in the Trichoderma-tomato interaction. The expression level of the Trichoderma acicularis CP3 (TasG_08790) gene (NCBI Gene ID: 36612810) was significantly upregulated by 9.9-fold at 9 hours of interaction, confirming it as a core effector protein in the interaction. (See below) Figure 1Phylogenetic analysis showed that CP3 is evolutionarily conserved within the Trichoderma genus, clarifying its Cerato-platanin (CP) family affiliation. (See...) Figure 2 a. Table 1.

[0022]

[0023] The CP3 encoding gene was cloned into the prokaryotic expression vector pMalc2X, and a recombinant expression plasmid was constructed and transformed into *Escherichia coli* strain BL21(DE3). After expression at 30℃ and 1 mM IPTG induction, the CP3 protein was purified by affinity chromatography, and its molecular weight was determined to be 68.12 kDa by SDS-PAGE electrophoresis. Figure 2 b.

[0024] Verification of protein secretion function: The secretory characteristics of CP3 were verified using a yeast signal sequence capture assay. The signal peptide sequence of CP3 was cloned and inserted into the yeast expression vector pSUC2 to construct the recombinant plasmid pSUC2-SPCP3; a positive control (pSUC2-SPAvr1b) and a negative control (empty vector pSUC2) were also set up. The three plasmids were transformed into YTK12 yeast competent cells, and positive clones were screened after culturing on CMD-W deficient medium at 30℃ for 3-5 days. Positive clones were picked and seeded into YPRAA medium containing 2 μg / mL antimycin A and then stained with TTC. The results showed that yeast in the pSUC2-SPCP3 group and the positive control group could grow and turn red, while the negative control group could not grow, confirming that CP3 is a signal peptide-dependent secretory protein. Figure 4 ; Subcellular localization of CP3 was detected using colloidal gold immunoelectron microscopy: Tomato MM seedling root tips were treated with 10 μg / mL CP3-His protein, fixed in 2.5% glutaraldehyde, dehydrated using a gradient method, embedded, and sectioned. The sections were then incubated with Anti-6×His-tagged monoclonal antibody, followed by transmission electron microscopy (TEM) observation showing that gold particles were concentrated within the tomato cell nuclei. Figure 5 .

[0025] 2. Construction of recombinant *Trichoderma echinococcus* strains overexpressing CP3: Genomic DNA was extracted from *Trichoderma echinococcus* DQ-1 using the CTAB method, and the CP3 coding gene was amplified using this DNA as a template. The amplified CP3 coding gene was ligated into a vector containing a strong gpdA promoter to construct an overexpression recombinant vector. The recombinant vector was introduced into wild-type *Trichoderma echinococcus* protoplasts via PEG-mediated protoplast transformation. After selection for HYG resistance at 350 μg / mL, gene insertion was identified by PCR, and expression levels were verified by RT-qPCR (using tef1 as an internal reference gene). Recombinant strains (OECP3-15, OECP3-63) with CP3 expression levels 3-4 times higher than wild-type strains were obtained. Simultaneously, CP3 knockout strains (ΔCP3-11, ΔCP3-13) and complementary strains (Δ / ΔCP3-18, Δ / ΔCP3-21) were constructed as controls. (See below) Figure 6 .

[0026] 3. Verification of disease resistance function: Exogenous protein treatment experiment: 4-leaf stage MM tomato seedlings were selected and divided into a control group (root irrigation with PBS buffer) and a treatment group (root irrigation with 3 μg / mL CP3 purified protein). Nine hours later, 5 mm diameter mycelial blocks of *Botrytis cinerea* were inoculated onto tomato leaves and cultured for 4 days in an incubator at 25℃, 90%±5% humidity, and a 12-hour light cycle. The lesion area was measured using ImageJ software. The lesion area in the treatment group was reduced by 71% compared to the control group (t-test, n=10). Figure 3 .

[0027] Recombinant strain treatment experiment: Four-week-old hydroponic MM tomato seedlings were selected and divided into Mock group (sterile water), WT group (wild-type Trichoderma echinosporum spore suspension), and overexpression group (OECP3-15 / 63 strain spore suspension, concentration 1×10⁻⁶). 6 CFU / mL, knockout group (ΔCP3-11 / 13 strain spore suspension), and complementary group (Δ / ΔCP3-18 / 21 strain spore suspension); after 9 hours, they were inoculated with *Botrytis cinerea* mycelial blocks, and the lesion area was measured after 4 days of culture. The results showed that the lesion area in the overexpression group was significantly smaller than that in the WT group, the lesion area in the knockout group was significantly larger than that in the WT group, and the disease resistance in the complementary group recovered to the WT level (ANOVA and Tukey's HSD, n=10, see...). Figure 7 .

[0028] Verification experiment on different tomato varieties: MT tomato seedlings at the 4-leaf stage were selected and divided into a control group (PBS buffer) and a treatment group (3 μg / mL purified CP3 protein). Nine hours later, the seedlings were inoculated with *Botrytis cinerea* mycelial blocks. After 4 days of culture, the lesion area was measured. The lesion area in the treatment group was significantly reduced compared to the control group (ANOVA and Tukey's HSD, n=10), demonstrating that CP3 protein also has disease resistance effects in different tomato varieties. (See...) Figure 8 .

[0029] 4. Data statistics: All experiments were conducted with at least three biological replicates. Data are expressed as mean ± standard deviation. One-way ANOVA combined with Tukey's HSD multiple comparison test was used for comparisons among multiple groups. Independent samples t-test was used for comparisons between two groups. P < 0.05 was considered statistically significant. Data charts were generated using GraphPad Prism software.

[0030] The results showed that the purified protein of CP3, a Cerato-platanin (CP) family protein derived from *Trichoderma echinococcus* DQ-1, or *Trichoderma echinococcus* strains overexpressing CP3, applied via root irrigation (preferably at the four-leaf stage or four-week hydroponic stage of tomato), significantly enhanced the resistance of tomatoes to gray mold without adversely affecting tomato growth. The recombinant strain exhibited superior stability in practical applications, providing a new approach for the green control of tomato gray mold.

[0031] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0032] Trichoderma acicularis ( Trichoderma asperellum Cerato-platanin protein CP3 gene cDNA sequence: ATGCCTTCCTTCAGCTCTTCTCTCGGCTTCCTCGCCACCGCCGCTGCCACCGTCTCGGCCGTCGTCCTCCCCCGAGGCGGCTCCGTCTCCGTCACCCCCCACGAGCAATACTCGTCATCCATCGGTGTCCTCGGCTGCCACATCAACACCAACCGTGTCGCCTACTGGCCCGGAGCTGTCGACTGCAACAACATCTGCGTCAAGCTCACCTACCAGGGCCGCTCCCTCAACGTCCTCAAGATCGACTCTTCCGGCGGCGCCCACGACATCTCCTACGATGCCTGGAACACGCTCGTCTTCGGCCAGTCCGCCACCGCCGACCCCCAGCAGGGCGGCGGCTACACCATGGACTGGGAATTCGTCGACAACAGCCAGTGCGCCGACCTGCTGTACAACGGCAAGCTGCCTCTCAGCGCTGCCAACAGCGTCAACTATGTTGCCAGCTGCCTTCAGCAGCCCAACTCTTTTGCGGCTCAGAACTATGAGTTCATCAACATCCTCGATCCCGTGTGCCACTACGGTTATGACGAGGTCTGTTCCTTCAACCTGGCTGTGAGCAACCAGCCCAGCTGCCCTCACCAGCTCGGTGTCCCCAACACTCCCACCGGAGTGTCTGTCCAGAACATCCAGTATGGCACTGGTCTTGTCTACACTGCTTAA Trichoderma asperellum Trichoderma asperellum Amino acid sequence of Cerato - platanin protein CP3: MPSFSSSLGFLATAAATVSAVVLPRGGSVSVTPHEQYSSSIGVLGCHINTNRVAYWPGAVDCNNICVKLTYQGRSLNVLKIDSSGGAHDISYDAWNTLVFGQSATADPQQGGGYTMDWEFVDNSQCADLLYNGKLPLSAANSVNYVASCLQQPNSFAAQNYEFINILDPVCHYGYDEVCSFNLAVSNQPSCPHQLGVPNTPTGVSVQNIQYGTGLVYTA

Claims

1. Trichoderma acicularis ( Trichoderma asperellum The application of CP3, a Cerato-platanin (CP) family protein derived from [source name missing], in enhancing plant resistance to gray mold is characterized by [feature missing]. The plant is tomato, and the gray mold is caused by Botrytis cinerea (Glaucus spp.). Botrytis cinerea The CP3 protein has an amino acid sequence corresponding to the accession number XP_024756165.1, and its encoding gene accession number is 36612810 (Gene ID).

2. The application according to claim 1, characterized in that, The application method is to apply CP3 protein exogenously to tomatoes, or to inoculate tomatoes with Trichoderma acicularis strains expressing CP3 protein.

3. The CP3 protein according to claim 1, characterized in that, The protein belongs to the Cerato-platanin (CP) family, contains a conserved CP domain, has a signal peptide, is a secreted protein, and can be located in the nucleus of tomato cells.

4. A method for enhancing the resistance of tomatoes to gray mold, characterized in that, Includes the following steps: Apply CP3 protein at a concentration of 3 μg / mL to tomato roots, or inoculate tomato roots with a concentration of 1×10⁻⁶. 6 A suspension of Trichoderma spores overexpressing CP3 protein at CFU / mL.

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