CN116987162A - Caknot1-U5 protein and application thereof - Google Patents
Caknot1-U5 protein and application thereof Download PDFInfo
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- CN116987162A CN116987162A CN202310694716.5A CN202310694716A CN116987162A CN 116987162 A CN116987162 A CN 116987162A CN 202310694716 A CN202310694716 A CN 202310694716A CN 116987162 A CN116987162 A CN 116987162A
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- phytophthora capsici
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- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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Abstract
The invention discloses a CaKnot1-U5 protein and application thereof. According to the invention, the phytophthora capsici effector NLP779 is firstly discovered to induce the capsicum to generate the antibacterial peptide, structural domain enrichment analysis is carried out on the upregulation protein in TMT histology results, the Knot1 antibacterial peptide is discovered to be upregulated and expressed, and the Knot1 (namely the Caknot1-U5 protein) after exogenous purification is added into zoospores of phytophthora capsici, so that the activity of the zoospores, the elongation of bud tubes and the formation of attached spores can be inhibited. And after the phytophthora capsici effector NLP779 is sprayed on pepper plants, the pepper immune response can be caused, and the infection of the phytophthora capsici is inhibited. NLP779 and Caknot1-U5 can be used together to prevent and treat phytophthora capsici better.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a Caknot1-U5 protein and application thereof.
Background
Phytophthora capsici (Phytophthora capsici) can infect various solanaceae and cucurbitaceae crops such as peppers, tomatoes and the like to cause destructive diseases, is difficult to prevent and treat, seriously affects the quality and the yield of vegetables, and often causes huge economic loss. At present, chemical pesticides are mainly relied on for preventing and treating the disease, but the drug resistance of pathogenic bacteria is improved due to the large amount of chemical agents, and pesticide residues and environmental pollution are aggravated. The development of novel green plant protection methods is unprecedented.
Phytophthora capsici can secrete a large amount of effector agents when infecting a host, and can interfere with host immune response or change metabolic pathways through interaction with various targets in the host, thereby promoting infection and colonization of the host. However, if recognized by host-receptor proteins, effector agents activate host immune responses, limiting the spread of pathogenic bacteria. Therefore, the effector is a bridge and medium in the interaction process of the pathogenic bacteria and the host, can determine the virulence strength of the pathogenic bacteria, can regulate and control whether the plant resistance is excited or not, has important significance in function and action mechanism research, and can provide theoretical basis for designing a durable and efficient disease prevention and control strategy based on the interaction relationship of the pathogenic bacteria and the host.
Disclosure of Invention
The invention aims to provide a CaKnot1-U5 protein and application thereof.
To achieve the object of the present invention, in a first aspect, the present invention provides a CaKnot1-U5 protein (derived from capsicum), which is:
(A) A protein consisting of the amino acid sequence shown in SEQ ID NO. 4;
(B) And the protein which is derived from the (A) and has the same function and is obtained by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 4.
In a second aspect, the present invention provides a fusion protein which is a CaKnot1-U5 protein comprising a pro-lytic tag;
Wherein the pro-lytic tag may be selected from MBP (maltose binding protein), SUMO (small molecule ubiquitin-like modification protein), GST (glutathione transferase), TRX (thioredoxin), etc.
Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO. 6 (containing a TRX tag).
In a third aspect, the invention provides a nucleic acid molecule encoding the Caknot1-U5 protein or the fusion protein.
In a fourth aspect, the invention provides biological materials comprising the nucleic acid molecules, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineering bacteria, or transgenic cell lines.
In a fifth aspect, the invention provides the use of the CaKnot1-U5 protein or fusion protein and a prokaryotic or eukaryotic expression system for expressing the CaKnot1-U5 protein or fusion protein in inhibiting Phytophthora capsici and plant diseases caused by Phytophthora capsici.
Further, the application includes:
(1) Preparing the Caknot1-U5 protein or the fusion protein into a protein solution, or applying escherichia coli bacterial solution or dilution thereof for expressing the protein or the fusion protein into soil or seedling culture matrix around plant rhizosphere; or alternatively, the first and second heat exchangers may be,
(2) Preparing the Caknot1-U5 protein or the fusion protein into a protein solution, or carrying out root irrigation treatment on plants by using escherichia coli bacterial liquid or dilution liquid thereof for expressing the protein or the fusion protein; or (b)
(3) Preparing the CaKnot1-U5 protein or the fusion protein into a protein solution, or soaking the escherichia coli bacterial solution expressing the protein or the fusion protein or a dilution thereof; or (b)
(4) Preparing a protein solution by using the Caknot1-U5 protein or the fusion protein, or spraying an escherichia coli bacterial solution expressing the protein or the fusion protein or a dilution thereof on plants.
In a sixth aspect, the invention provides a composition comprising the CaKnot1-U5 protein or fusion protein, and phytophthora capsici effector NLP779 or a truncate thereof; or alternatively, the process may be performed,
the composition comprises a prokaryotic or eukaryotic expression system for expressing the Caknot1-U5 protein or fusion protein and a prokaryotic or eukaryotic expression system for expressing phytophthora capsici effector NLP779 or a truncated form thereof.
Wherein, phytophthora capsici leonian effector NLP779 is:
(a) A protein consisting of the amino acid sequence shown in SEQ ID NO. 1;
(b) And (b) a protein which is derived from (a) and has equivalent functions and is obtained by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 1.
The phytophthora capsici effector NLP779 truncated body consists of 54 th-277 th amino acids of a sequence shown in SEQ ID NO. 1.
The nucleotide sequence of the NLP779 encoding gene is shown as SEQ ID NO. 2.
In a seventh aspect, the present invention provides the use of the composition for inhibiting phytophthora capsici and plant diseases caused by phytophthora capsici.
Further, the application includes:
(1) Preparing the composition into a solution and applying the solution to soil around plant rhizosphere or seedling substrate; or alternatively, the first and second heat exchangers may be,
(2) Preparing the composition into a solution to irrigate roots of plants; or (b)
(3) Preparing the composition into a solution for seed soaking treatment; or (b)
(4) Plants were sprayed with the composition formulated as a solution.
In the present invention, the plants include, but are not limited to, capsicum, arabidopsis thaliana.
According to the invention, the phytophthora capsici effector NLP779 is firstly discovered to induce the capsicum to generate the antibacterial peptide, structural domain enrichment analysis is carried out on the upregulation protein in TMT histology results, the Knot1 antibacterial peptide is discovered to be upregulated and expressed, and the Knot1 (namely the Caknot1-U5 protein) after exogenous purification is added into zoospores of phytophthora capsici, so that the activity of the zoospores, the elongation of bud tubes and the formation of attached spores can be inhibited. And after the phytophthora capsici effector NLP779 is sprayed on pepper plants, the pepper immune response can be caused, and the infection of the phytophthora capsici is inhibited. NLP779 and Caknot1-U5 can be used together to prevent and treat phytophthora capsici better.
Drawings
FIG. 1 shows the expression of NLP779 at various stages of Phytophthora capsici life history in a preferred embodiment of the invention.
FIG. 2 shows NLP779 domain prediction in a preferred embodiment of the invention.
FIG. 3 shows the nucleotide and amino acid sequences of NLP779 in a preferred embodiment of the invention.
FIG. 4 shows NLP779PCR amplification results in a preferred embodiment of the invention.
FIG. 5 shows NLP779 induced cell death in N.benthamiana cells (expected size of each protein, protein size markers in kDa, detected by Western blotting using GFP) in a preferred embodiment of the invention.
FIG. 6 shows NLP779 gene silencing in accordance with a preferred embodiment of the invention. (A) Detecting silencing efficiency (< P < 0.001) of the silencing transformant by RT-qPCR; (B) growth rate of WT and silent transformant, scale bar 1cm; (C) WT versus silencing transformant diameter statistics (ns P > 0.05).
FIG. 7 is a NLP779 silencing transformant virulence assay according to a preferred embodiment of the invention. (A) WT and silencing transformant infects pepper leaves for 60h (under ultraviolet lamp), and the scale is 1cm; (B) Diameter statistics of infected pepper leaf spot (ns P > 0.05); (C) Infection of pepper leaves qPCR detects phytophthora capsici biomass (ns P > 0.05).
FIG. 8 shows NLP779 gene overexpression in the preferred embodiment of the invention. (A) RT-qPCR detects the silencing efficiency of the over-expressed transformant (P < 0.001); (B) growth rates of WT and overexpressing transformants, scale bar 1cm; (C) WT vs overexpressing transformant diameter statistics (ns P > 0.05).
FIG. 9 is a NLP779 over-expressed transformant virulence assay according to a preferred embodiment of the invention. (A) The scale of the WT and the overexpressing transformant in a state of infecting pepper leaves for 60 hours (under ultraviolet) is 1cm; (B) Spot diameter statistics of infected pepper leaves (< P < 0.001); (C) Infection of pepper leaves qPCR detects phytophthora capsici biomass (< P0.01).
FIG. 10 is a graph showing secretion verification of NLP779 over-expressed transformants in the preferred embodiment of the present invention.
FIG. 11 shows a hydrophilic assay of NLP779 protein according to a preferred embodiment of the invention. The horizontal axis represents the amino acid number of the protein, the vertical axis represents hydrophobicity, and the larger the protein represents hydrophobicity, the more negative the protein represents hydrophilicity.
FIG. 12 shows NLP123779-53 protein purification in accordance with a preferred embodiment of the invention. (A) NLP779-53 PCR amplification; (B) SDS-PAGE (control: no IPTG added; 6 positive transformants after induction with T1-T6 plus IPTG) (C) SDS-PAGE; (D) a gel filtration exclusion chromatography mAU graph.
FIG. 13 shows the trypan blue staining after injection of NLP779 protein 4d at different concentrations in the preferred embodiment of the invention.
FIG. 14 shows NLP779 protein inhibits phytophthora capsici infection in a preferred embodiment of the invention. (A) 1 mu MNLP779 and the proportion of phytophthora capsici infected with capsicum leaf 60h (under ultraviolet) after PBS treatment is 1cm; (B) leaf spot diameter statistics (×p < 0.001); (C) qPCR detects relative biomass of phytophthora capsici in leaves (< 0.001 by P).
FIG. 15 is a graph showing NLP779 induced active oxygen burst in pepper leaves in accordance with the preferred embodiment of the present invention. (A) DAB dyeing results, black scale 1cm, white scale 100 μm; (B) hydrogen peroxide content of capsicum leaves; (C) RT-qPCR detection of CaRbohA transcript levels.
FIG. 16 shows NLP779 protein induced callose deposition in Arabidopsis thaliana, according to a preferred embodiment of the present invention, with a scale of 100. Mu.m.
FIG. 17 shows NLP779 protein activating phosphorylation of pepper MAPKs in a preferred embodiment of the invention.
FIG. 18 shows analysis of heat map of ethylene synthesis and response gene expression after NLP779 treatment and verification of expression level by RT-qPCR in the preferred embodiment of the invention.
FIG. 19 shows the analysis of the heat map of jasmonic acid synthesis and response gene expression after NLP779 treatment and the verification of the expression level by RT-qPCR in the preferred embodiment of the invention.
FIG. 20 is a thermal map analysis of the response of the phenylpropane metabolic pathway to gene expression after NLP779 treatment and a PAL enzyme activity assay according to a preferred embodiment of the present invention.
FIG. 21 shows up-regulated protein domain enrichment after NLP779 treatment in accordance with a preferred embodiment of the present invention.
FIG. 22 shows Caknot1-U5 RT-qPCR validation after NLP779 treatment in accordance with a preferred embodiment of the present invention.
FIG. 23 is an alignment of amino acids A0A2G2YZQ8 and A0A2G2YZU5 in a preferred embodiment of the invention.
FIG. 24 shows prokaryotic expression and purification of CaKnot1-U5 in a preferred embodiment of the invention. (A) PCR amplification of Caknot 1-U5; (B) SDS-PAGE (control: no IPTG added; 6 positive transformants after induction with T1-T6 plus IPTG) (C) SDS-PAGE; (D) a gel filtration exclusion chromatography mAU graph.
FIG. 25 shows the inhibition of Phytophthora capsici by Caknot1-U5 in a preferred embodiment of the invention. (A) Percentage of zoospore activity after treatment with different concentrations of CaKnot1-U5 (B and C) CaKnot1-U5 and germination rate after treatment with PBS (< 0.001, <0.0001, < P), in a scale of 50 μm.
FIG. 26 shows that Caknot1-U5 and NLP779 have more remarkable effect of inhibiting phytophthora capsici infection when being applied cooperatively in the preferred embodiment of the invention. (A) Plant states after 3d and 5d infection of phytophthora capsici under different treatments; (B) The infested state (under ultraviolet) of part of the leaves under different treatments; (C) qPCR verifies the phytophthora capsici biological quantity of each group.
Detailed Description
Pc123779 (Coordinates: 51:306813-307446) is cloned from phytophthora capsici LT1534 genome, named NLP779, and the function and action mechanism of the Pc123779 are primarily studied, and the main results are as follows:
(1) NLP779 plays a role in early and mid-stage infection: inducing cell necrosis in Nicotiana benthamiana overexpressing NLP 779; the transcription level of the phytophthora capsici is detected in the growth and infection stage, and the RT-qPCR result shows that the transcription level does not change obviously in the mycelium, sporangium, zoospore and germinating spore stages of the phytophthora capsici, and the expression is up-regulated in 6h of infection, which indicates that the phytophthora capsici plays a role in early and middle stages of infection.
(2) NLP779 is an effector of the non-classical secretory pathway: NLP779 is predicted to have no classical signal peptide, but has non-classical pathway secretion characteristics. NLP779 was detected in Phytophthora capsici broth by Westen Blot; and after onion transiently expresses NLP779-GFP, it is separated by a plasma wall, and green fluorescence is observed outside the cell membrane to indicate that the onion can be secreted outside the cell, and contrast GFP is only positioned in the cell membrane. We therefore infer that NLP779 is an effector secreted by a non-classical pathway.
(3) NLP779 is a virulence factor: silencing NLP779 has no significant influence on phytophthora capsici pathogenicity and overexpression enhances the phytophthora capsici pathogenicity, which indicates that NLP779 is a virulence factor, and that silencing has no significant influence may not have significant influence on phytophthora capsici pathogenicity by singly silencing one NLP due to the functional redundancy relationship between different NLPs.
(4) NLP779 can activate plant immunity: exogenous application of NLP779 protein can stimulate plant immunity including active oxygen burst, callose deposition, MAPKs phosphorylation and inhibit phytophthora capsici infection.
(5) NLP779 regulates the multiple hormonal pathways of capsicum: and TMT (TandemMass Tags) proteomics analysis is carried out after NLP779 is externally applied to the pepper, compared with a control group, the NLP779 is enriched in a large amount of up-regulated expressed proteins in a jasmonic acid pathway, an ethylene pathway and a phenylpropane metabolic pathway after being treated, and RT-qPCR transcription level verification is carried out on part of genes, so that the result is consistent with TMT histology, and the NLP779 can regulate and control pepper immunity through the above pathways.
(6) NLP779 induces capsicum to produce antibacterial peptide: the structural domain enrichment analysis is carried out on the up-regulated protein in the TMT histology result, and the discovery is that the Knot1 antibacterial peptide is up-regulated in expression, and the exogenous purified Knot1 is added into zoospores of Phytophthora capsici, which can inhibit the activity of the zoospores, the elongation of the bud tubes and the formation of attached spores.
The results of the study indicate that NLP779 is an atypical secretory effector, which is both a virulence factor and an elicitor, and has potential for development as a plant immune elicitor because it can effectively activate a host immune response.
Materials used in the following examples:
1. plant material and strain: the Nicotiana benthamiana (Nicotiana benthamiana) is a laboratory preservation and planting method, the Capsicum (Capsicum annuum) is a laboratory preservation and selfing line 06221 (see documents: chen Shanshan, ai Congcong, cheng Hui and the like), the phytophthora capsici effector RxLR19781 is one-to-one verified with the yeast interaction of the interaction protein thereof [ J ]. Shandong university of agriculture (Nature science edition), 2019,50 (01): 44-48), and the Arabidopsis thaliana (Arabidopsis thaliana) seeds are a laboratory preservation method. The planting conditions are 22 ℃ and the humidity is 65 ℃, and the planting is carried out in a light-dark alternating greenhouse for 14h/10 h.
The cloning strain of the escherichia coli is Escherichia coliDH5 alpha, and the expression strain is BL21 (DE 3), rosetta (DE 3) and Rosetta-gamiB; agrobacterium strain GV3101 is purchased from Quan Shi gold and Optimaceae; phytophthora capsici (Phytophthora capsici) strain LT1534 was kept by the present laboratory.
2. Test carrier: the vector pET28a (+), pET21b (+), and pET32a used for prokaryotic expression are stored in the laboratory. The agrobacterium-mediated transient expression vectors pBIN-GFP2 and pFag, and the oomycete pTOR expression vector are given away by Nanjing university of agriculture plant protection institute.
3. Experimental primers: the primers used for prokaryotic expression purification were designed using DNAMAN software, and RT-qPCR primers were designed in part using the Primerquest (https:// sg. Idtdna. Com/pages/tools/primer questreturnurl =% 2FPrimerquest%2FHome%2 FIndex) website and the primers used for ligation to the pBINeGFP vector were designed for the Norvezan webpage (https:// crm. Vazyme. Com/cetool/simple. Html).
The primer sequences used in the following examples are shown in Table 1.
TABLE 1
The experimental methods involved in the following examples:
1. extracting genomic DNA of the plant and phytophthora capsici: extraction was performed using the convalet century CW0553S kit.
2. Extracting RNA of leaf blades and phytophthora capsici: extraction using the sparkeasylplant rnakitac0305 kit.
3. RNA was reverse transcribed into cDNA and manipulated according to conventional methods.
4. Gene cloning and plasmid construction
4.1 amplification of DNA fragments, PCR reaction system was as follows:
F：5′-CGCGGATCCATGACCGACAGTAAAAACACC-3′
R：5′-CCGCTCGAGTTTTTTTTCGCCAAATGG-3′
the mixture of the system is placed in a PCR instrument, the obtained PCR product is subjected to agarose gel electrophoresis, a band which is consistent with the size of the target gene is cut and retrieved, and the band is purified by using a OMEGAGel ExtractionKit kit.
4.2 Gene and vector cleavage and recovery
The double enzyme digestion reaction system is as follows:
the above system was mixed and then digested in a 37℃water bath for 45-60min, and 360. Mu.L of binding buffer was added to the digested product. The purification steps are the same as above.
4.3 recombinant plasmid ligation and E.coli transformation
The connection system is as follows:
the above system was mixed and placed in a 16 ℃ metal joint overnight.
(1) Taking out the competent E.coli, melting on ice, adding the connection product into 50. Mu.L of competent E.coli, ice-bathing for 30min, water-bathing for 90s at 42 ℃, transferring to ice-bathing for 2min, adding 500. Mu.LLB liquid culture medium into each tube, and shake culturing at 37 ℃ for 45-60min.
(2) Taking out the recovered Escherichia coli, centrifuging at 6000rpm for 1min, discarding part of supernatant, re-suspending and precipitating with residual LB, diluting and coating on LB plate with corresponding resistance, and culturing in a 37 deg.C constant temperature incubator for 16-20 hr.
(3) The monoclonal is selected in a centrifuge tube added with 1mLLB, and shake cultured for 4-6h at 37 ℃.
(4) And (3) performing PCR verification by taking the bacterial liquid as a template, sequencing the monoclonal bacterial liquid with correct bands, and preserving the monoclonal bacterial liquid after error-free sequencing.
4.4 recombinant plasmid extraction, operating according to conventional method.
5. The recombinant plasmid transformed with Agrobacterium was manipulated according to the conventional method.
6. Agrobacterium-mediated transient expression
(1) Resuscitate the stored Agrobacterium solution overnight for expansion culture, centrifuge at 3800rpm for 5min, discard supernatant, and add 10mLMgCl 2 The solution, resuspended pellet, was repeated three times.
(2) Every 100mLMgCl 2 2mL of pH5.7 MES and 20. Mu.L of acetosyringone were added to the solution.
(3) The sediment is resuspended by the buffer solution, the OD of the bacterial liquid is regulated to 0.5, and the bacterial liquid is placed in an incubator at 28 ℃ for culturing for 4 hours.
(4) And selecting proper benthamiana or capsicum leaves, injecting the agrobacterium suspension into the benthamiana or capsicum leaves from the back surfaces of the leaves, and minimizing damage to the leaves.
7. WesternBlot (WB), according to the usual methods.
8. Trypan blue staining, DAB staining and callose staining
(1) Trypan blue staining: the harvested plant material was placed in 100mL of pre-heated trypan blue dye solution, boiled water bath for 90s (prolonged time as appropriate depending on the dyeing situation). And (3) after the dyeing is completed, continuously dyeing for 24 hours, and then replacing the chloral hydrate for decoloration, wherein the chloral hydrate is replaced for 24 hours until the decoloration is completed. Placed in 95% alcohol, photographed and recorded.
(2) DAB staining: the collected plant materials are placed in DAB for dyeing, transferred to 95% alcohol for boiling water bath for decolorization after 24 hours of shading dyeing, and photographed and recorded.
(3) Callose staining: placing the harvested plant material in lactophenol and absolute ethanol 2:1, decoloring in a water bath at 65 ℃ in the mixed solution until the leaves are nearly transparent, taking out the leaves, washing the leaves by deionized water, placing the leaves in an aniline blue dye solution for dyeing overnight, taking out the leaves, placing the leaves in a deionized water horizontal shaking table for 2min for 3 times, and placing the leaves in a fluorescence microscope for observation and photographing after making a sample.
9. PAL crude enzyme liquid extraction and enzyme activity determination
(1) Extracting PAL crude enzyme liquid: the treated plant material was placed in a centrifuge tube, placed in two steel balls, quick frozen with liquid nitrogen, ground in a grinder, added with 3mL borate buffer (100 mM, pH 8.8) per 250mg, mixed with 1mM EDTA and 5% insoluble polyvinylpyrrolidone (PVPP), and vortexed to mix well. Centrifuging at 12500rpm for 10min, and obtaining supernatant as PAL crude enzyme solution.
(2) PAL enzyme activity assay: 600mL of crude enzyme solution, 250mL of phenylalanine (20 mM) and 1mL of borate buffer (100 mM, pH 8.8) were incubated at room temperature for 1h, and 100. Mu. LHCl (6N) was added to terminate the reaction. PAL activity was determined by measuring the OD of trans-cinnamic acid formed at 290 nm. The calculation formula is as follows:
PAL Activity [ U/(gFW.h)]＝(A 290 ×vt×V)/(0.01×Vs×FW×t)
vt: total enzyme solution volume (mL); FW: fresh weight of leaf (g); vs: measuring the amount (mL) of the enzyme solution; v: total volume of reaction solution (mL); t: reaction time (h).
10. Prokaryotic expression protein
The specific steps are as follows: zhao Li structural features of RxLR effector and RxLR145 regulatory mechanism explore [ D ]. Shandong university of agriculture, 2018.
11. Recombinant protein purification
11.1 affinity chromatography
The specific steps are as follows: zhao Li structural features of RxLR effector and RxLR145 regulatory mechanism explore [ D ]. Shandong university of agriculture, 2018.
11.2 gel exclusion filtration chromatography
The specific steps are as follows: zhao Li structural features of RxLR effector and RxLR145 regulatory mechanism explore [ D ]. Shandong university of agriculture, 2018.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: aLaboratyManual, 2001), or as recommended by the manufacturer's instructions.
EXAMPLE 1NLP779 high expression in early and mid stages of Phytophthora capsici infection
Total RNAs of two types of stages of phytophthora capsici are extracted respectively, namely, a growing stage such as hypha (Mycelia, MY), sporangium (SP), zoospores (Zoospore, ZO) and germinating spores (Germinating Cysts, GC), and the zoospores of phytophthora capsici infect the total RNAs of 1.5h, 3h, 6h, 12h, 24h, 48h, 72h and 108h of pepper leaves, and the result shows that the transcript level of the RT-qPCR detection part NLPs is not obviously up-regulated in the growing development stage of phytophthora capsici, but up-regulated in the early stage of the infection stage to about 6h up-regulated to the maximum multiple, and the NLP779 possibly does not participate in the vegetative growth stage of phytophthora capsici but plays an important role in the early and middle stages of infection (fig. 1).
Example 2NLP779 bioinformatics analysis
1. Gene sequence analysis
NLP779 (Coordinates: 51:306813-307446) is 844bp in length, encodes 277 amino acids, 29.67kDa, two cysteines at positions 93 and 119, with NPP1 conserved domain at positions 77-270 amino acids as analyzed by NCBI domain.
2. Predicting NLP779 to be free of signal peptide but secreted by a non-classical secretory pathway
Predicted by SignalIP-5.0 (https:// services. Heathttech. Dtu. Dk/services. Phphttps:// services.healthtech.dtu.dk/service.phpSecretomeP-1.0)And 2.0 (https:// services. Healthcare. Dtu. Dk/se)rVICE. PhpSeccetomeP-2.0) predicted its secretion by a non-classical secretory pathway, NN-score of 0.612 and Secp-score of 0.876462, both greater than a threshold of 0.6.
3. Prediction and modeling of NLP779 secondary and tertiary structure
Secondary structure prediction was performed using psipred (http:// bioif. Cs. Ucl. Ac. Uk/psipred).
NLP779 three-level structure modeling is performed by using SWISS-MODEL (https:// swissmodel. Expasy. Org /), 6QBE, 3GNU and 5NNW are selected for modeling according to website sequence analysis, and GMQE values are 0.70, 0.65 and 0.64,QMEANDisCo Global and 0.87+/-0.006, 0.78+/-0.006 and 0.77+/-0.006 respectively, so that the 6QBE structure is finally selected as a final homologous modeling object. According to modeling results, NLP779 is consistent with classical NLP, the core conserved region is in the form of a beta-sheet sandwich with two sides and three opposite sides, and a disulfide bond exists.
The nucleotide and amino acid sequences of NLP779 are shown in FIG. 3.
EXAMPLE 3NLP779 Gene cloning and vector construction
cDNA of phytophthora capsici strain LT1534 is used as a template, DNAMAN is used for designing a specific amplification primer of NLP779, and pBIN-GFP2 is selected as a carrier. The amplified products are subjected to agarose gel electrophoresis analysis, gel recovery and purification are carried out, double enzyme digestion is carried out on the amplified products and the vector plasmid respectively, DH5 alpha is connected and converted to construct recombinant plasmid, bacterial colony PCR verification is carried out, gene sequencing is carried out on the recombinant plasmid by the engineering company of the Qinke biology, and the vector fragment and the target gene sequence are compared to be completely consistent (figure 4).
Example 4NLP779 is capable of inducing Orthosiphon cell death
The successfully constructed NLP779 recombinant plasmid is transformed into GV3101 agrobacterium competent, NLP779 is inoculated into the leaf of Benshi tobacco by using a method of agrobacterium-mediated transient expression, INF1 (NCBI accession number: XM_ 002900382.1) is used as a positive control, GFP is used as a negative control, buffer is used as a blank control, necrosis is observed after 4d inoculation, and a photograph is taken, and trypan blue staining is used for remarkable visualization. Both NLP779 and INF1 showed necrosis, and neither GFP nor Buffer had necrosis. Immunoblot analysis showed that all recombinant proteins were correctly expressed in the leaf of nikovia tubes at the expected size (fig. 5).
EXAMPLE 5 silencing of NLP779 in Phytophthora capsici
1. Silencing NLP779 does not affect the vegetative growth rate of Phytophthora capsici
Gene silencing NLP779 is carried out by RNAi technology, 300bp is selected to reversely construct in pTOR vector (NLP 779 silencing fragment is shown as SEQ ID NO: 3), and PEG-mediated protoplast transformation is used to obtain the phytophthora capsici transformant silencing NLP 779. RT-qPCR performed NLP779 transcriptional level verification. Two positive transformants were co-screened for about 70% silencing efficiency (P < 0.001) for ST4 and ST7 (fig. 6A), and the remaining transformants were either near WT or less efficient to silence without affecting phytophthora capsici growth rate (fig. 6, b and C). No significant change in ST2 transcript levels (P > 0.5) was used as a control for the subsequent experiments.
The growth rate of NLP779 silent transformants was measured, ST4, ST7, ST2 and phytophthora capsici LT1534 (WT) were grown simultaneously in NPB medium, in the dark in an incubator at 25℃for 2d and recorded by photographing, and the results showed that the colony growth conditions of the silent transformants ST4, ST7 and the control group ST2, WT were about the same (P > 0.5), indicating that silencing NLP779 had no significant effect on the vegetative growth rate of phytophthora capsici.
2. Silencing NLP779 has no obvious influence on phytophthora capsici infection virulence
To verify the difference of the pathogenicity of the silent transformants compared with that of the WT to the host, the above ST4, ST7, ST2 and WT were respectively perforated with a 0.4cm diameter puncher by selecting new mycelia from the outer edge of the colonies, the mycelia were inoculated down to the same sites of approximately balanced growing pepper leaves, and infection was observed and photographed at 60 h. The results showed that the diameter of infection of ST4, ST7 was about the same as ST2 and WT with no significant change (P > 0.05) (fig. 7, a and B), total DNA was extracted from the corresponding leaves and qPCR was performed to detect phytophthora capsici biomass (fig. 7C), and the results were also about the same (P > 0.05), indicating that silencing NLP779 had no significant effect on phytophthora capsici infection. EXAMPLE 6 overexpression and secretion verification of NLP779 in Phytophthora capsici
1. Overexpression of NLP779 does not affect the vegetative growth rate of Phytophthora capsici
The method is the same as the silencing of NLP779, the full length of NLP779 is constructed on a phytophthora capsici expression vector in the forward direction, the transcription level change of NLP779 is detected by RT-qPCR, two positive transformants OT7 and OT9 are screened, the transcription level of NLP779 is respectively improved to about 60 times and 200 times (P < 0.001) (figure 8A), the transcription level of OT3 is not significantly changed and WT is used as a control group (P > 0.05), and in addition, the growth conditions of the transformants over expressing NLP779 and the control group are not significantly different (P > 0.05) (figures 8, B and C), so that the over-expression of NLP779 in phytophthora capsici has no obvious influence on the growth of the transformants.
2. Overexpression of NLP779 enhances virulence of Phytophthora capsici
In order to verify the difference of pathogenicity of the over-expressed transformant relative to that of WT on a host, the above OT7, OT9, OT3 and WT were respectively perforated with a 0.4cm diameter puncher by selecting new mycelia on the outer edge of the colony, the mycelia were inoculated down to the same parts of the approximately balanced growing pepper leaves, and infection was observed and photographed at 60 h. The results showed that there was no significant difference in the spot diameters of OT3 and WT as control (P > 0.05), the OT7, OT9 infection diameters were greater than control (P < 0.001) (fig. 9, a and B), indicating that over-expression of NLP779 in phytophthora capsici enhanced virulence in phytophthora capsici, while extracting the corresponding leaf extract total DNA and performing qPCR to detect phytophthora capsici biomass, OT7 and OT9 phytophthora capsici biomass were also greater than control (P < 0.01) (fig. 9C).
3. NLP779 can be secreted from Phytophthora capsici to extracellular
About 10 square blocks of 0.3cm×0.3cm were cut out of the NPB plates using a scalpel for burning and sterilizing WT, OT7, OT9, OT3, and grown in NPB liquid medium, and after 4d, the growing hyphae protein extract was taken out and subjected to SDS-PAGE and WB, and detected with his antibody. The culture broth was first filtered through 4 layers of gauze, the filtrate was concentrated in a 10kDa protein ultrafiltration tube, SDS-PAGE and WB were performed, and actin proved not to be secreted as a loading group control. The OT7 and OT9 hypha antibodies were both expressed, and the OT7 and OT9 were also expressed in the concentrated culture broth, with actin having an expression band in the hypha protein sample, and no in the concentrated culture broth, indicating that NLP779 was secreted into the culture medium without destroying the hypha, indicating that it was an exocrine effector (FIG. 10).
EXAMPLE 7 prokaryotic expression and purification of NLP779
1. NLP779 prokaryotic expression and purification
DNAMAN designs a specific amplification primer of NLP779, and the vector selects PET-28a for fusion expression with His-tag.
NLP779 is mostly in the middle of precipitation with little soluble protein. Optimizing the purification conditions, such as increasing or decreasing IPTG concentration, increasing or decreasing induction temperature, changing Buffer type and PH during purification, had no significant effect, indicating that the full length NLP779 recombinant protein appears as inclusion bodies in e.coli, and the supernatant was almost free of soluble proteins, so the supernatant could not be subjected to nickel column affinity chromatography. Dialysis after precipitation using urea, less active protein after renaturation, and therefore, truncations are considered.
2. NLP779 truncate design, prokaryotic expression and purification
By combining predicted secondary and tertiary structures, truncations (consisting of amino acids 54-277 of the sequence shown in SEQ ID NO: 1) are designed without breaking the core region and without cleaving disulfide bonds. Analysis of NLP779 amino acid sequence hydrophobicity shows that the first 50 amino acids are more hydrophobic, which is unfavorable for soluble expression of protein. Epasy analysis predicted that NLP779 Instability Index (II) was classified as a labile protein as 46.40. Average hydrophilicity (GRAVY) index-0.378. Epasy analysis predicted Instability Index (II) to be 25.30 classified as a stable protein after the first 53 hydrophobic region amino acids were removed. Average hydrophilicity (GRAVY) index-0.477, both hydrophilicity and stability were improved after truncation (fig. 11).
And (3) redesigning the primer, amplifying and constructing the primer into a PET-21b carrier, and transferring the plasmid into escherichia coli Rosette competence after sequencing and comparison, namely successful connection. After successful expression, the supernatant was subjected to ultrasonication, nickel column affinity chromatography and sampling at each stage after extensive culture to show that the content of soluble protein was significantly increased, and high purity protein was obtained after gel exclusion filtration chromatography (FIG. 12) for subsequent functional experiments, and buffer A was used in the purification process (buffer A was prepared by weighing Hepes 14.30g and NaCl 35.06g, respectively, and ultrapure water was fixed to a volume of 2L and dissolved sufficiently, and was placed in a refrigerator at 4℃overnight, and then pH was adjusted to 7.5, and filtered by vacuum pump using a water-based filter membrane of 0.22. Mu.m).
Example 8 functional verification of NLP779 recombinant protein
1. NLP779 causes necrosis of Benshi smoke and capsicum cells
To verify whether the purified protein is an active protein, the following experiment was designed. The purified NLP779 truncated protein was inoculated into leaf discs of Nicotiana benthamiana, and could cause necrosis of Nicotiana benthamiana cells, and only required a concentration of about 50nM (FIG. 13). NLP779 recombinant protein is inoculated to pepper leaves, so that leaf wilting and necrosis are caused rapidly, and part of leaves fall off and are more severe than the Nicotiana benthamiana. The results prove that the purified protein is active protein and can induce necrosis of Nicotiana benthamiana and capsicum leaves.
2. Spray application of NLP779 for inhibiting phytophthora capsici infection
Since capsicum leaf necrosis is remarkable and leaf shedding occurs, it is suggested that the plant cell allergic necrosis reaction (Hypersensitive Response, HR) is also rapid necrosis, and it is hypothesized that HR may be generated at the inoculation site. NLP779 truncated protein with concentration of 1 mu M is sprayed on pepper plants, and PBS buffer solution is sprayed on a control group. And inoculating phytophthora capsici blocks after 2 hours, observing the infection condition of the phytophthora capsici after 60 hours, photographing and recording, measuring the diameter, respectively extracting DNA, and performing qPCR to detect the biomass of the phytophthora capsici.
After NLP779 protein is sprayed, the diameter of the lesion is smaller than that of a control group, and the qPCR results are consistent. The results show that the pepper can inhibit phytophthora capsici infection after the NLP779 truncated protein is sprayed (figure 14).
3. NLP779 stimulates the accumulation of active oxygen in capsicum
Reactive oxygen species (Reactive oxygen species, ROS) are highly reactive defense molecules that are rapidly produced by plants after infection with microorganisms, often resulting in oxidative stress. In plants, ROS production is primarily dependent on peroxidases or plasma membrane bound NADPH oxidases. Plants spontaneously produce active oxygen when subjected to biotic and abiotic stresses, mainly including hydrogen peroxide, superoxide anions, and the like. ROS are responsible for regulating multiple signaling pathways, including immune responses to pathogens, HR, and Kong Guanbi, among others. Hydrogen peroxide as one of the important components of active oxygen can be reflected in its accumulation in plants by DAB staining.
DAB staining was performed after spraying NLP779 recombinant protein and PBS buffer at a concentration of 1. Mu.M in capsicum respectively for 2 hours, and it was found that the capsicum leaves after NLP779 treatment were observed to be darker brown than the control group. In addition, pepper leaves before treatment, namely 0h and 0.5h, 2h, 4h, 12h, 24h and 48h after treatment are respectively collected, and the hydrogen peroxide content in the leaves is quantitatively detected by a titanium sulfate colorimetric method, so that the pepper leaves have obvious accumulation compared with a control group. RNA of the experimental group and the control group are extracted in different time periods, and the RT-qPCR detection of the active oxygen marker gene CaRbohA is carried out, and the transcription level of the active oxygen marker gene CaRbohA is improved by about 10 times compared with that of the control group CaRbohA after 1.5h of NLP779 treatment. The experimental results in fig. 15 show that NLP779 is capable of inducing the burst and accumulation of active oxygen in capsicum.
4. NLP779 induces the deposition of callose in Arabidopsis thaliana
When plants are infected or stressed by pathogenic bacteria, callose synthesis increases, preventing bacterial invasion. The aniline blue staining method can be used for callose reactions. The samples were taken for aniline blue staining after 48h using 0.1 μm, 1 μm NLP779 truncate recombinant protein and PBS buffer, respectively, and the leaves after NLP779 treatment produced a more pronounced fluorescent signal compared to the control treatment, and callose accumulation increased with increasing concentration of NLP779 (fig. 16).
5. NLP779 activates MAPKs phosphorylation of capsicum annuum
Activation of the MAPK cascade is an early signaling event for PTI and ETI, followed by modulation of immune output, including hormone production and transcriptional reprogramming, etc. Activation of the MAPK cascade generally involves sequential phosphorylation of MAPK kinase, MAPK kinase and MAPK, which plays a key role in plant resistance to pathogenic bacteria. In this experiment, after 1. Mu.M NLP779 was sprayed with capsicum, samples were taken at 15min, 30min, and 60min, respectively, and the MAPK phosphorylation level was reflected by using the phosphorylation antibody p 44/42. The results showed that the mapk phosphorylated protein started to respond 15min of NLP779 treatment and the phosphorylation level was significantly elevated for 30min compared to the control. This suggests that NLP779 mediates downstream defense responses by activating MAPK signaling pathways (fig. 17).
EXAMPLE 9 tandem mass spectrometry proteomic analysis
Peppers were inoculated with NLP779 truncate protein and PBS buffer, respectively, and after 12h were sampled and subjected to tandem mass spectrometry (TMT) proteomic analysis, and 36451 Peptides (Peptides) were identified by spectral analysis, wherein specific Peptides (Unique Peptides) were 32914, and 7067 proteins were identified in total, of which 5985 were quantifiable. The comparison group is significantly up-regulated according to the relative quantitative value being more than 1.3 times or less than 1/1.3, and the differential expression protein is obtained by screening the statistical T test p <0.05, and significantly down-regulated according to the relative quantitative value being more than 1.3. There were 372 proteins significantly down-regulated and 445 proteins significantly up-regulated.
Ethylene, jasmonic acid, salicylic acid, etc. are important hormones for plants, and the active level of such pathways can indirectly reflect stress-resistant levels of plants. Thus protein enrichment was performed for such pathways.
1. Ethylene pathway related protein enrichment
Ethylene has been previously found to promote fruit ripening and is well known. In addition, it is an important plant hormone which plays an important role in plant growth and development and biological abiotic stress. Activation of the ethylene pathway-related enzymes is therefore often able to exert positive regulatory effects against pathogen infection. Through enrichment of TMT proteomics upregulation proteins, protein levels of CaACS and CaERF gene families were found to be upregulated, and additionally ACC synthase was a precursor for ethylene synthesis, therefore, caACC and CaERF14 were selected for transcription level verification by RT-qPCR, and compared with control, the CaACC synthase of the pepper leaves treated with NLP779 had upregulation expression from 1.5h to 48h, and CaERF14 was returned to normal level after rapid upregulation within a short period of 1.5h and 3h, indicating that NLP779 might activate the pepper ethylene pathway to enhance pepper disease resistance (FIG. 18).
2. Jasmonate pathway-related protein enrichment
Jasmonic acid is also used as an important hormone for plants, can induce the secondary growth of the plants, and can regulate the insect resistance and disease resistance of the plants. Can also induce the synthesis of components such as anthocyanidin, ketone, alkaloid and the like in plants, promote the gene expression of the dissolving ferment and inhibit the growth of bacteria. Through enrichment of TMT proteomics upregulation proteins, jasmonic acid pathway-related gene CaLOX, caAOS, caAOC, caOPR2 up-regulates expression and jasmonic acid pathway antagonistic protein CaJAZs down-regulates expression. In addition, pdf1.2 is also a classical marker gene of the jasmonic acid pathway, so that CaPdf1.2 and CaOPR2 were selected for RT-qPCR for transcriptional level verification. Compared with the control, the expression of CaPdf1.2 and CaOPR2 of the capsicum sprayed with NLP779 is up-regulated. The results indicate that NLP779 may enhance pepper immunity by activating the pepper jasmonic acid pathway (fig. 19).
3. Phenylpropane metabolic pathway related protein enrichment
Phenylpropane metabolism is one of important secondary metabolic pathways of plants, and metabolites thereof, such as lignin, sporopollen, anthocyanin, organic acid, and the like, play an important role in regulating plant adaptive growth. Lignin, one of the metabolites, resists invasion of pathogenic bacteria, resists feeding of herbivores, participates in resisting abiotic stress and the like, so that the improvement of the activity of enzymes related to the phenylpropane metabolic pathway can improve the resistance of plants to pathogenic substances and provide a physical barrier for the plants. Enrichment of TMT-group phenylpropane metabolic pathway related proteins we found elevated levels of CaPAL, ca4CL, caHCT proteins. PAL phenylalanine ammonia lyase as the most important enzyme in this pathway, its activity directly reflects the activity level of this pathway. After NLP779 treatment, we extracted crude enzyme liquid of PAL, phenylalanine as substrate, compared with control, PAL enzyme activity of the capsicum leaf after NLP779 treatment is significantly higher than that of control group, reaches the highest enzyme activity at about 24h, and is improved by about 4 times compared with control. The results indicate that NLP779 may increase pepper immunity by increasing the activity of the phenylpropane metabolic pathway (fig. 20).
4. Upregulated protein domain enrichment
By domain enrichment of up-regulated proteins after treatment of NLP779 truncations (FIG. 21), the families of alkene terpene synthases, cytochrome P450, pathogenic related proteins, bet v1, ABC transporters, etc. up-regulate expression, in addition, we found proteins of interest, gamma-thionin family proteins, which are plant antibacterial peptides, are shorter by about 10kDa and are rich in cysteines, with disulfide bonds in their interior imparting their extremely high chemical, thermal and proteolytic stability. According to the basic division of the domains of the antibacterial peptide into thionins, defensins, hevein-like peptides, knottin-typepeptides, lipidtransferproteins and alpha-hairpinin and snakins family, the enriched 4 antibacterial peptides are Knottin (Knot 1) domain proteins. The Knot1 family protein has antibacterial activity, kills digestive enzymes in insects and even has inactivation to cancer cells, so that the Knot1 protein with high activity has very strong application value, can be developed into green agriculture and pesticides, and can even play a certain role in treating cancers.
TMT histology was enriched to 4 Knot1 family proteins (Table 2) altogether, with A0A2G2YZA, A0A2G2XJD without signal peptide predicted to be localized to plant chloroplasts and mitochondria, respectively, and A0A2G2YZQ8, A0A2G2YZU5 predicted to have signal peptide localized to the extracellular side, according to SignalIP predictions.
TABLE 2Caknot1 Gene profiling
We verified at the transcriptional level that the CaKnot1-U5 transcript levels of the pepper leaves after NLP779 truncate protein treatment were up-regulated, consistent with the trend of TMT proteomics results (FIG. 22).
EXAMPLE 10Caknot1 Gene function Studies
1. CaKnot1 Gene sequence analysis
The exosome space of the plant cell membrane is the first battlefield for plant defense pathogen infection, so A0A2G2YZQ8 and A0A2G2YZU5 with signal peptide are selected for further study. Q8 and U5 are 321bp, and code for 106 amino acids and 12kDa. The amino acid similarity was 81.13% and all contained 8 cysteines (FIG. 23).
2. Prediction of three-level structure of CaKnot1-U5 protein
Three-level structure prediction is carried out by using alphafold (https:// www.alphafold.ebi.ac.uk /), the core area is three reverse beta-sheets and one alpha helix, 8 cysteines in the core area form 4 internal disulfide bonds (the amino acid sequence of the Caknot1-U5 protein is shown as SEQ ID NO:4, and the nucleotide sequence of the coding gene is shown as SEQ ID NO: 5).
3. CaKnot1 prokaryotic expression and purification
CaKnot1-U5 is constructed in PET-28a and PET-21b prokaryotic expression vectors, and expression strains Rossette or BL21 are inclusion bodies. The main reason for the formation of inclusion bodies is hypothesized to be that many disulfide bonds are very prone to mismatch due to the strongly reducing environment in the large intestine. Thus, to ensure that the correct disulfide bond is formed between its cysteines, TRX is expressed in fusion with this protein. And the expression strain of Rosseta-gamiB is selected, the Caknot1-U5 fused with TRX expresses and the soluble protein is purified (the amino acid sequence of the Caknot1-U5 fused with TRX is shown as SEQ ID NO:6, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO: 7). The purification procedure used buffer B (buffer B was prepared by weighing Tris 7.26g and NaCl 70.2g, respectively, and ultra-pure water to a volume of 2L and dissolving thoroughly), placing in a refrigerator at 4deg.C overnight, adjusting pH to 8.5, and filtering with 0.22 μm water-based filter membrane by vacuum pump. The experimental results are shown in FIG. 24.
4. Caknot1 inhibits zoospore activity and spore germination of phytophthora capsici
Since the purified Knot1 carries a TRX tag of about 20kDa and itself has only about 10kDa, there is a fear of affecting the function thereof, and therefore, in order to prove that the purified protein is active, the following experiment was designed. The purified Knot1 protein is added into zoospores of phytophthora capsici to make the final concentration 10 mu M and 50 mu M, and PBS buffer solution is added as a control. Zoospores were active well before protein was added. After addition of protein, less than about half of the zoospores of the experimental group with a final concentration of 10. Mu.M did not swim. The zoospore activity of the experimental group with the final concentration of 50 mu M is obviously reduced, and almost no zoospores are swimming in the visual field. Zoospores of the PBS control group were not significantly different from before. The percentage of zoospores active in the visual field was counted (fig. 25A), and the inhibition rate of 10 μm was about 60% (p=0.0002), and the inhibition rate of 50 μm was about 90% (P < 0.0001). The purified protein is shown to be active, and simultaneously, the Caknot1-U5 can obviously inhibit zoospore activity of phytophthora capsici.
Zoospores of the experimental group and PBS control group with final concentration of 50. Mu.M were transferred onto new V8 medium, respectively, and germination status was observed after 4 hours (FIG. 25B), only a small part of zoospores of the 50. Mu.M experimental group were germinated (P < 0.0001), and the length of the sprout tube was smaller than that of the Mock group. Knot1 was shown to be able to inhibit the germination of zoospores (fig. 25C).
5. The NLP779 and the Caknot1 are matched for use, so that the effect of inhibiting phytophthora capsici infection is more remarkable
NLP779 truncated protein can actively activate capsicum to generate a defense reaction so as to resist infection of phytophthora capsici, and CaKnot1 can directly inhibit activity of phytophthora capsici, so that NLP779 and CaKnot1 are matched for use, and three experimental groups and a blank control group are set. Selecting capsicum with approximately the same growth vigor, spraying 1 mu MNLP779 protein for 2 hours by using a Co-use group, spraying zoospores of phytophthora capsici into the capsicum, spraying 50 mu M Knot1 protein for 30 minutes, and spraying Knot1 protein for the second time at intervals of 2 hours. Knot1 group replaced NLP779 with PBS. NLP779 group then replaced Knot1 with PBS. Mock group replaced all proteins with PBS.
And respectively spraying zoospores of phytophthora capsici for 24h, 48h and 72h, collecting partial total DNA extracted from the capsicum leaves, and performing qPCR (quantitative polymerase chain reaction) detection on the biomass of the phytophthora capsici. And 3d and 5d, observing, photographing and recording the whole capsicum. And when 5d, the main stems of the Mock group peppers are necrotic and collapse wilted due to phytophthora capsici infection. The leaf was picked and observed under ultraviolet light to see the severity of infection, with NLP779 and Co-use groups being less symptomatic. The qPCR results showed that the biomass of Phytophthora capsici in the Co-use group was smaller than that in the other groups, indicating that NLP779 used in combination with Caknot1 was better able to control Phytophthora capsici (FIG. 26).
Studies have shown that NLP779 is a virulence factor of Phytophthora capsici, and when sprayed on pepper plants, it can induce a pepper immune response and inhibit infection of Phytophthora capsici. It was verified that it stimulated plant immunity by ROS level detection, callose deposition, MAPKs phosphorylation, etc. Numerous hormones in plants such as ethylene, jasmonic acid, salicylic acid, and the like also regulate plant immunity. After the NLP779 is externally applied, TMT proteomics analysis is carried out, and compared with a control group, the TMT proteomics analysis is enriched to a large amount of up-regulated expression proteins in an ethylene pathway, a jasmonic acid pathway and a phenylpropane metabolic pathway, which shows that the TMT proteomics analysis regulates plant immunity through multiple pathways. Experimental results indicate that NLP779 serves as both a virulence factor and a dual identity of the exciton.
NLP779 stimulates the capsicum to produce a kind of Knot1 antibacterial peptide, and researches show that part of Knot1 has antibacterial, disease and pest resistant and anticancer cell activities, and can open up a new direction for reducing the use of antibiotics and novel anticancer drugs. After prokaryotic expression and purification, relevant experiments on phytophthora capsici activity are carried out, which shows that the phytophthora capsici can inhibit zoospore activity of the phytophthora capsici and spore germination rate and bud tube length, and the invention does not carry out relevant experiments on insect pest resistance and tumor cell activity on Caknot 1-U5. NLP779 destroys cell membrane by binding to GIPC on dicotyledon cell membrane surface to cause cell membrane instantaneous perforation, knot1 destroys cell membrane by binding to PA and PA congener on fungi and bacteria membrane surface, GIPC and PA head are all glucosamine, NLPs are secreted weapon of pathogenic bacteria, knot1 is secreted weapon of killing pathogenic bacteria of plant, they all select sphingolipid substance on cell membrane surface as target point, it shows importance and conservation of sphingolipid substance on pathogenic bacteria and plant cell membrane, sphingolipid is important component of biological membrane system, it is also key signal molecule necessary for controlling cell internal environment stabilization, adapting stress and regulating plant immunity, participate in plant cell death and defense reaction, but how to sense pathogen and transduction signal during interaction of plant pathogen, especially micro domain in sphingolipid in plasma membrane is still to be explored in future study.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
- A caknot1-U5 protein characterized in that it is:(A) A protein consisting of the amino acid sequence shown in SEQ ID NO. 4;(B) And the protein which is derived from the (A) and has the same function and is obtained by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 4.
- 2. A fusion protein, characterized in that it is a CaKnot1-U5 protein comprising a pro-lytic tag;wherein the dissolution promoting tag is selected from MBP, SUMO, GST, TRX;the Caknot1-U5 protein is as described in claim 1.
- 3. The fusion protein of claim 2, wherein the amino acid sequence is as shown in SEQ ID NO. 6.
- 4. A nucleic acid molecule encoding the protein of claim 1 or the fusion protein of claim 2 or 3.
- 5. A biological material comprising the nucleic acid molecule of claim 4, wherein the biological material is recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector or an engineering bacterium.
- 6. Use of the protein of claim 1 or the fusion protein of claim 2 or 3 and a prokaryotic or eukaryotic expression system expressing said protein or fusion protein for inhibiting phytophthora capsici and plant diseases caused by phytophthora capsici.
- 7. The application according to claim 6, characterized in that it comprises:(1) Preparing the protein or the fusion protein into a protein solution, or applying escherichia coli bacterial liquid or dilution thereof for expressing the protein or the fusion protein into soil or seedling culture matrixes around plant rhizosphere; or alternatively, the first and second heat exchangers may be,(2) Preparing the protein or the fusion protein into a protein solution, or irrigating roots of plants by using escherichia coli bacterial liquid or dilution liquid thereof for expressing the protein or the fusion protein; or (b)(3) Preparing the protein or the fusion protein into a protein solution, or soaking the escherichia coli bacterial liquid or the dilution liquid thereof for expressing the protein or the fusion protein; or (b)(4) Preparing a protein solution by using the protein or the fusion protein, or spraying an escherichia coli bacterial solution or a dilution thereof for expressing the protein or the fusion protein on plants.
- 8. A composition comprising the protein of claim 1 or the fusion protein of claim 2 or 3, and phytophthora capsici effector NLP779 or a truncate thereof; or alternatively, the process may be performed,The composition comprises a prokaryotic or eukaryotic expression system for expressing the protein of claim 1 or the fusion protein of claim 2 or 3, and a prokaryotic or eukaryotic expression system for expressing phytophthora capsici effector NLP779 or a truncate thereof;wherein, phytophthora capsici leonian effector NLP779 is:(a) A protein consisting of the amino acid sequence shown in SEQ ID NO. 1;(b) A protein derived from (a) with equivalent functions and with one or more amino acids substituted, deleted or added by the sequence shown in SEQ ID NO. 1;the phytophthora capsici effector NLP779 truncated body consists of 54 th-277 th amino acids of a sequence shown in SEQ ID NO. 1.
- 9. Use of the composition of claim 8 for inhibiting phytophthora capsici and plant diseases caused by phytophthora capsici.
- 10. The application according to claim 9, characterized in that it comprises:(1) Preparing the composition into a solution and applying the solution to soil around plant rhizosphere or seedling substrate; or, (2) preparing the composition into a solution to perform root irrigation treatment on plants; or (b)(3) Preparing the composition into a solution for seed soaking treatment; or (b)(4) Plants were sprayed with the composition formulated as a solution.
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