Hrpx protein plant disease resistance peptide and application thereof

Abstract

Hrpx protein plant disease resistance peptide and application thereof relate to a plant disease resistance peptide and application thereof.The Hrpx protein plant disease resistance peptide is Hrpx-5, has plant disease resistance, and the amino acid sequence of Hrpx-5 is shown as SEQ ID NO:1.The application of the Hrpx protein plant disease resistance peptide as a plant disease resistance activator, the Hrpx protein plant disease resistance peptide Hrpx-5 is an active ingredient in the plant disease resistance activator.The necrosis area of tobacco leaves treated by the Hrpx protein plant disease resistance peptide Hrpx-5 of the application is obviously larger than that of the Hrpx protein, and the tobacco leaves can also induce obvious HR response under low concentration.The Hrpx protein plant disease resistance peptide of the application is superior to the full-length Hrpx protein, and has higher thermal stability.

Description

Technical Field

[0001] This invention relates to plant disease-resistant peptides and their applications. Background Technology

[0002] Harpin proteins are a class of proteins belonging to a protein family, mostly found in Gram-negative bacteria. They were first discovered by Dr. Wei Zhongmin in *Brwinia amylovora*, a Gram-negative bacterium, and are encoded by the *hrp* gene. Current research indicates that these proteins are relatively stable at high temperatures, exhibiting thermostability; they readily react with some proteases, such as proteinase K; and they are rich in glycine, with some Harpin proteins containing small amounts of cysteine. Besides inducing pathogenic responses in susceptible host plants, and triggering allergic reactions and growth-promoting effects in resistant and non-host plants, Harpin proteins can also induce many desirable plant traits, such as increased yield and quality, and enhanced plant immunity. Summary of the Invention

[0003] The purpose of this invention is to provide Hrpx protein plant disease-resistant peptides with superior performance compared to full-length Harpin proteins.

[0004] The plant disease resistance peptide Hrpx-5 of this invention has plant disease resistance; the amino acid sequence of Hrpx-5 is shown in SEQ ID NO: 1.

[0005] The application of Hrpx protein plant disease resistance peptide as a plant disease resistance activator, with Hrpx-5 being the active ingredient in the plant disease resistance activator.

[0006] The necrotic area of ​​tobacco leaves treated with the Hrpx protein plant disease-resistant peptide Hrpx-5 of this invention was significantly larger than that of the Hrpx protein itself, and it could also induce a significant HR response in tobacco leaves at low concentrations. Hrpx-5 reached its peak value faster than the full-length Hrpx protein, and the peak H2O2 content of the peptide Hrpx-5 was significantly higher than that of Hrpx. No obvious lesions were found in tobacco leaves treated with Hrpx-5. The ratio of lesion area to tobacco leaf area for both Hrpx and Hrpx-5 was significantly smaller than that for the negative control (**P<0.01), and the ratio of lesion area to tobacco leaf area for Hrpx-5 was significantly smaller than that for tobacco leaves treated with Hrpx (**P<0.01). The Hrpx protein plant disease-resistant peptide of this invention is superior to the full-length Hrpx protein and has greater thermal stability. Attached Figure Description

[0007] Figure 1This is a diagram showing the analysis results of the amino acid sequence of the Hrpx protein using the ExPASy protein hydrophilicity / hydrophobicity prediction tool in Specific Implementation Method 1; where amino acid residues above the threshold line are hydrophobic and amino acid residues below the threshold line are hydrophilic.

[0008] Figure 2 This is a diagram showing the results of online transmembrane region analysis of the Hrpx protein using TMHMM Server in Specific Implementation Method 1.

[0009] Figure 3 This is a diagram showing the results of the online prediction of the secondary structure of the Hrpx protein using NPS@:SOPMA secondary structure prediction in Implementation Method 1.

[0010] Figure 4 This is a predicted diagram of the coiled helix structure of the Hrpx protein according to the present invention;

[0011] Figure 5 This is a predicted diagram of the tertiary structure of the Hrpx protein according to the present invention;

[0012] Figure 6 This is the predicted Hrpx protein signal peptide diagram of the present invention;

[0013] Figure 7 This is a predicted phosphorylation site map of the Hrpx protein in this invention;

[0014] Figure 8 This is a graph showing the results of the Hrpx protein activity assay of this invention;

[0015] Figure 9 This is a graph showing the experimental results of Hrpx and Hrpx-5 peptide-induced HR response activity assay in Example 2;

[0016] Figure 10 This is a graph showing the experimental results of the HR response induced by Hrpx, Hrpx-B, Hrpx-5 and Hrpx-5B in Example 3;

[0017] Figure 11 These are the main experimental parameters for determining the H2O2 content in tobacco leaf tissue in Example 4;

[0018] Figure 12 These are the experimental data from Example 4 regarding the determination of H2O2 content in tobacco leaf tissue, including the oxygen burst induced by Hrpx, Hrpx-B, Hrpx-5, and Hrpx-5B.

[0019] Figure 13 This refers to the H2O2 content in tobacco leaves at different times after different Harpin treatments, as determined in Example 4.

[0020] Figure 14 This is a graph showing the resistance of tobacco pretreated with purified Hrpx, Hrpx-B, Hrpx-5, and Hrpx-5B to TMV in Example 5; where, Figure 14 -b represents the ratio of the lesion area to the tobacco leaf area;

[0021] Figure 15 This is a subcellular localization diagram of Agrobacterium on tobacco, as shown in Example 5. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0024] Specific implementation method one: The Hrpx protein in this implementation method has the amino acid sequence shown in SEQ ID NO: 2.

[0025] The Hrpx protein in this embodiment is composed of 137 amino acids, has a relative molecular mass of 13631.48, a theoretical isoelectric point of 4.06, and a molecular formula of C0.05. 557 H 883 N 175 O 215 S5 has a total of 1835 atoms. Among the 137 amino acids in the Hrp protein, glycine (Gly), glutamine (Gln), serine (Ser), and leucine (Leu) are abundant, accounting for 20.40%, 15.30%, 13.90%, and 7.30% of the total amino acids, respectively. There are four positively charged arginine (Arp) and lysine (Lys) amino acids, and eleven negatively charged aspartic acid (Asp) and glutamate (Glu). Because the Hrpx protein is rich in glycine and contains only one cysteine ​​residue, it has high thermal stability. The end of the sequence is glutamine (Gln). Its half-life in mammalian cells in vitro is 30 h; in yeast, it is >20 h; and in E. coli, it is >10 h.

[0026] The amino acid sequence of the Hrpx protein was analyzed using the ExPASy protein hydrophilicity / hydrophobicity prediction tool. The results are as follows: Figure 1As shown, the hydrophilic amino acids of the Hrpx protein are concentrated between positions 5-44 and 54-89, while the hydrophobic amino acids are concentrated between positions 44-53 and 89-102. The Hrpx protein as a whole is a hydrophilic protein. Its Arg score at position 60 is the lowest (-2.444), indicating the strongest hydrophilicity; Leu at position 49 and Ile at position 50 have the highest scores (1.889), indicating the strongest hydrophobicity.

[0027] The transmembrane region of the Hrpx protein was analyzed online using TMHMM Server, and the results are as follows: Figure 2 As shown, Hrpx protein has no transmembrane region and belongs to non-transmembrane protein, which cannot form a corresponding region with Hrpx affinity. This further confirms that Hrpx is a type of protein that is secreted by the type III secretion system.

[0028] The secondary structure of Hrpx was predicted online using NPS@:SOPMA secondary structure prediction; the analysis results are as follows: Figure 3 As shown, its random coils are displayed in yellow (55.47%), α-helices in blue (29.93%), β-turns in green (8.03%), and β-sheets in red (6.57%). It can be seen that Hrpx has a large number of random coils. The α-helices of the Hrpx protein account for 29.93%, concentrated in amino acids at positions 39-54 and 90-104.

[0029] The coil-and-coil structure (CC domain) of the Hrpx protein was analyzed online using COILS Server software. The results are as follows: Figure 4 As shown, the results indicate that there is only one possible coiled-coil structure for the Hrpx protein, located at positions 41-54. Combined with the prediction of the secondary structure of the Hrpx protein above, it was found that the coiled-coil structure of the Hrpx protein overlaps with the α-helix region.

[0030] The SWISS-MODEL database was used to search for the template protein XPsB, which is similar to Hrpx, as a template for modeling. However, the similarity was low, only 48.15%. This is because the Hrpx protein contains a large number of random coils, which makes the analysis of its crystallization and tertiary structure extremely difficult. Therefore, there are no proteins in the database with the resolved 3D structures of proteins similar to Hrpx. The resulting tertiary structure prediction model is as follows: Figure 5 As shown, random coils are the predominant secondary structure, while distinct α-helical structures are also visible. Random coils account for 55.47% of the total secondary structures, and α-helices account for 29.93%, mainly concentrated in amino acids at positions 39-54 and 90-104.

[0031] Signal peptide prediction of the Hrpx protein amino acid sequence was performed online using Signal 4.1 software. The results are as follows: Figure 6 As shown, the C, S, and Y values ​​of Hrpx protein are all less than 0.5, which is below the threshold line. Therefore, it is indicated that Hrpx protein has no signal peptide and is a type of protein that does not require a signal peptide to exert its effects. This is consistent with the actual situation, as Hrpx protein itself is a type of protein that acts directly on the cell interior by relying on the type III secretion system.

[0032] The phosphorylation sites of the Hrpx protein were predicted and analyzed using NetPhos 3.1. The results are as follows: Figure 7 As shown, the Hrpx protein is subject to phosphorylation modification, with serine having the most phosphorylation sites and threonine having only one possible phosphorylation site.

[0033] Experiments showed that the Hrpx protein could only maintain its tertiary structure, activity, and stability when the buffer solution consisted of 50 mmol / L Tris, 0.9% NaCl, 20% glycerol, and a pH of 7.5, with a denaturation temperature of 86.9℃.

[0034] The optimal storage temperature for Hrpx protein is 4°C. At 4°C, Hrpx protein does not show significant degradation, and only a small amount of protein remains in the precipitate. At -80°C, although Hrpx protein does not show significant degradation, the amount of protein in the precipitate increases significantly. At 22°C, Hrpx protein shows significant degradation, therefore it is not suitable for storage.

[0035] Specific Implementation Method 2: The Hrpx protein plant disease resistance peptide described in this implementation method is Hrpx-5, which has plant disease resistance; the amino acid sequence of Hrpx-5 is shown in SEQ ID NO: 1; the molecular weight is 2264.64, and the isoelectric point is 5.83.

[0036] SEQ ID NO: 1Hrpx protein plant disease resistance peptide showed good solubility in buffer solution (buffer solution composition: 50 mmol / L Tris, 0.9% NaCl, 20% glycerol and pH 7.5), with a solubility concentration generally ≤2 mg / mL or ≤5 mg / mL.

[0037] Example 1

[0038] The activity of Hrpx protein was experimentally determined in tobacco and Arabidopsis thaliana, with PD 10 buffer as a negative control. A certain concentration of Hrpx protein was diluted 10-fold and 100-fold and injected into tobacco leaves; the presence of an HR response was observed after 24 hours. Arabidopsis thaliana seeds were soaked in diluted Hrpx protein and cultured on MS medium for 7 days; root length was then measured. Diluted Hrpx protein was sprayed onto Arabidopsis thaliana, and the aboveground growth was observed.

[0039] The activity and growth-promoting function of the purified Hrpx protein were verified by measuring its expression in tobacco and Arabidopsis thaliana, respectively. The results are as follows: Figure 8 As shown, Figure 8 -a shows that obvious allergic reactions occurred at the injection sites of Hrpx protein and Hrpx protein diluted 10 times on tobacco leaves, while no changes were observed in the negative control. Figure 8 -b shows that the root length of Arabidopsis thaliana treated with Hrpx protein increased significantly compared with the negative control, with a root length of about 14.44 mm, while the root length of the negative control was about 10 mm. Figure 8 -c shows that the aboveground parts of Arabidopsis thaliana sprayed with Hrpx protein grew more vigorously than the negative control, and the results were the same for all four replicate experiments.

[0040] In summary, the purified Hrpx protein is active and can induce the HR response in tobacco leaves. It also has growth-promoting functions, promoting root growth and aboveground growth in Arabidopsis thaliana.

[0041] Example 2

[0042] The prepared Hrpx protein and peptide Hrpx-5 were diluted with PD 10 buffer to concentrations of 10 μM, 1 μM, and 0.1 μM, respectively, and then aliquoted into sterile EP tubes. The protein was slowly injected into mesophyll cells from the underside of the leaf using a syringe at appropriate injection sites on tobacco leaves, and the extent of protein infection was marked with a marker. The HR response of the leaves was observed approximately 48 hours later. PD 10 buffer was used as a negative control, and three replicates were performed on three leaves of similar growth from the same tobacco plant, with each experiment repeated three times.

[0043] result:

[0044] Hrpx and Hrpx-5 induced a significant HR response in tobacco leaves, while the negative control did not induce an HR response (e.g., Figure 9 (As shown in the figure). It can also be seen that the necrotic area of ​​tobacco leaves treated with Hrpx-5 is significantly larger than that of Hrpx protein, and it can also induce a significant HR response in tobacco leaves at low concentrations, while Hrpx only has a slight HR response at a concentration of 0.01 μM.

[0045] Example 3

[0046] Fully expanded tobacco leaves were pretreated by spraying with 10 μM proteins Hrpx, Hrpx-B (Hrpx was diluted to 10 μM with PD 10 buffer and aliquoted into sterilized EP tubes; one group was subjected to heat treatment by boiling in a water bath for 5 min), Hrpx-5, and Hrpx-5B (Hrpx-5 was diluted to 10 μM with PD 10 buffer and aliquoted into sterilized EP tubes; one group was subjected to heat treatment by boiling in a water bath for 5 min). After 20 h of pretreatment, the prepared injection solution was injected into the entire leaf. PD 10 buffer served as a negative control. After 5 days, the percentage of lesion area to the total leaf area represented the disease incidence. The lesion area and total leaf area were calculated using ImageJ software. The experiment was repeated three times, with three plants treated with each protein.

[0047] result:

[0048] Purified Hrpx (10 μM), heat-treated Hrpx-B (10 μM), Hrpx-5 (10 μM), and heat-treated Hrpx-5B (10 μM) were injected into tobacco leaves, respectively. PD 10 buffer was used as a negative control to observe the HR response induced in tobacco leaves. Unlike the negative control, Hrpx, Hrpx-B, Hrpx-5, and Hrpx-5B induced significant HR responses in tobacco leaves (e.g., ...). Figure 10 (As shown). The ratio of necrotic area to injection area of ​​Hrpx-5 was significantly greater than that of Hrpx (P<0.01), Hrpx-B (P<0.01), and Hrpx-5B (P<0.05). It was also found that the thermal stability of Hrpx-5 was significantly better than that of Hrpx protein. After high-temperature heat treatment, the reduction in the HR response of tobacco leaves induced by Hrpx was significantly greater than that of Hrpx-5.

[0049] Example 4

[0050] The prepared Hrpx protein and peptide Hrpx-5 were diluted with PD 10 buffer to a concentration of 10 μM and then aliquoted into sterile EP tubes. One group underwent heat treatment by boiling in a water bath for 5 minutes, while the other group was not heat-treated. Both were then sprayed evenly onto tobacco leaves until no droplets fell. At 0, 1, 3, 6, 9, and 12 hours after inoculation, the treated leaves were harvested, and two leaf discs were punched using a 1.5 cm diameter punch. PD 10 buffer was used as a negative control. Three leaves of similar growth from the same tobacco plant were selected to form three parallel groups, with each group's experiment repeated three times.

[0051] Using the H2O2 assay kit (Nanjing Jiancheng Bioengineering Institute), take two tobacco leaf discs treated with each of the above proteins, add 1 mL of PD 10 buffer, grind them into a juice in a mortar, centrifuge at low speed, and collect the supernatant for testing. The main experimental parameters are shown in (e.g.) Figure 11 (As shown).

[0052] result:

[0053] Purified Hrpx (10 μM), heat-treated Hrpx-B (10 μM), Hrpx-5 (10 μM), and heat-treated Hrpx-5 (10 μM) were uniformly sprayed onto tobacco leaves. The treated leaves were harvested at 0, 1, 3, 6, 9, and 12 h after treatment to measure their H2O2 content, with PD 10 buffer as a negative control. Unlike the negative control, the H2O2 content of tobacco treated with Hrpx, Hrpx-B, Hrpx-5, and Hrpx-5B showed significant changes (e.g., ...). Figure 12 and 13 (As shown in the image). It can be seen that peptides Hrpx-5 and Hrpx-5B reach their peak values ​​faster than the full-length proteins Hrpx and Hrpx-B, and the peak H2O2 content of Hrpx-5 is significantly higher than that of Hrpx. Hrpx-5 and Hrpx-5B reach their peak values ​​after 1 hour of treatment, with H2O2 contents of 191.5 mmol / L and 134.5 mmol / L, respectively. Hrpx and Hrpx-B reach their peak values ​​after 6 hours of treatment, with H2O2 contents of 145.3 mmol / L and 126.9 mmol / L, respectively. Simultaneously, it can be seen that high-temperature heat treatment has a significant impact on oxygen eruption; the peak H2O2 contents of Hrpx-B and Hrpx-5B are significantly lower than those of Hrpx and Hrpx-5.

[0054] Example 5

[0055] Agrobacterium (GV3101-TMV-GFP) was kindly provided by Professor Wu Huijun of Nanjing Agricultural University. Single colonies of Agrobacterium were picked and inoculated into 10 mL of LB broth containing Km+Rif, and cultured overnight at 28°C with shaking at 200 rpm. 2 mL of the overnight-cultured Agrobacterium was transferred to 100 mL of LB broth at a 2% inoculation rate and cultured at 28°C with shaking at 200 rpm until the OD 600 was between 0.8 and 1.0. The cells were harvested by centrifugation at 5000g for 15 min, and resuspended in a prepared resuspension solution to achieve a final OD 600 of 0.5. After incubation at room temperature for 2-3 hours, the cells were injected into tobacco.

[0056] Fully expanded tobacco leaves were pretreated by spraying with 10 μM proteins Hrpx, Hrpx-B (Hrpx was diluted to 10 μM with PD 10 buffer and aliquoted into sterilized EP tubes; one group was subjected to heat treatment by boiling in a water bath for 5 min), Hrpx-5, and Hrpx-5B (Hrpx-5 was diluted to 10 μM with PD 10 buffer and aliquoted into sterilized EP tubes; one group was subjected to heat treatment by boiling in a water bath for 5 min). After 20 h of pretreatment, the prepared injection solution was injected into the entire leaf. PD 10 buffer served as a negative control. After 5 days, the percentage of lesion area to the total leaf area represented the disease incidence. The lesion area and total leaf area were calculated using ImageJ software. The experiment was repeated three times, with three plants treated with each protein.

[0057] Epidermal cells were extracted from tobacco leaves that developed lesions after being injected with Agrobacterium and the subcellular localization of Agrobacterium in the tobacco leaves was observed using a laser confocal microscope.

[0058] result:

[0059] Twenty hours before TMV inoculation, purified Hrpx (10 μM), heat-treated Hrpx-B (10 μM), Hrpx-5 (10 μM), and heat-treated Hrpx-5 (10 μM) were uniformly sprayed onto tobacco leaves, with PD 10 buffer as a negative control. It was observed that, compared to the negative control, the disease index and the number of lesions were significantly reduced in the leaves treated with the protein. No obvious lesions were found in the tobacco leaves treated with Hrpx-5 (e.g., ...). Figure 14 (As shown in -a). The ratio of lesion area to tobacco leaf area for Hrpx, Hrpx-B, Hrpx-5, and Hrpx-5B was significantly smaller than that of the negative control (**P<0.01), and the ratio of lesion area to tobacco leaf area for Hrpx-5 was significantly smaller than that of tobacco leaves treated with Hrpx (**P<0.01). The proportion of lesion area of ​​Hrpx-B after high-temperature heat treatment increased significantly compared with Hrpx (*P<0.05), while the lesion area of ​​Hrpx-5B after high-temperature heat treatment also increased compared with Hrpx-5, but the change was not significant.

[0060] Epidermal cells from tobacco leaves with lesions were extracted, and their fluorescence positions were observed using a laser confocal microscope. The results showed... Figure 15 As shown, a relatively obvious green fluorescence appears at 475 nm, which is concentrated in the intercellular space. Therefore, after Agrobacterium is transformed in tobacco, its subcellular localization is in the intercellular space.

Claims

1. Hrpx protein plant disease-resistant peptide, characterized in that, The Hrpx protein plant disease resistance peptide is Hrpx-5, which has plant disease resistance; the amino acid sequence of Hrpx-5 is shown in SEQ ID NO:

1.

2. The application of the Hrpx protein plant disease-resistant peptide according to claim 1 in improving plant disease resistance, characterized in that, The plant in question is tobacco, and the disease is tobacco mosaic virus.

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