Cape1 peptide segment of c-terminal of ptpr1 protein and application thereof

Abstract

This invention discloses the CAPE1 peptide at the C-terminus of the PtPR1 protein and its applications. There are three types of CAPE1 peptides in the PtPR1 protein: PtPR1f (amino acid sequence shown in SEQ ID NO.1), PtPR1j (amino acid sequence shown in SEQ ID NO.2), and PtPR1l (amino acid sequence shown in SEQ ID NO.3). Experimental results show that the three CAPE1 peptides at the C-terminus of the PtPR1 protein provided by this invention can induce an immune defense response in woody plants at low concentrations. The use of these three CAPE1 peptides can also significantly improve the disease resistance of woody plants to *Colletotrichum gloeosporioides*, *Pseudomonas aeruginosa*, *Botrytis cinerea*, or *Phytophthora spp.*, without inhibiting the growth of these pathogens. Therefore, it can be applied to the prevention and control of woody plant diseases, providing a new approach to improving the disease resistance of woody plants and showing broad application prospects in the green control of woody plant diseases.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the CAPE1 peptide at the C-terminus of the PtPR1 protein and its applications. Background Technology

[0002] In recent years, with the continuous expansion of poplar plantation areas and the extension of continuous cropping periods, disease problems have become increasingly serious, posing a bottleneck to the healthy development of the poplar industry. Therefore, exploring effective measures for the prevention and control of poplar diseases is of great significance for promoting the sustainable development of the poplar industry.

[0003] Pathogenesis-Related Protein 1 (PR1), induced by pathogen invasion of host plants, plays a central role in plant disease resistance, inducing systemically acquired resistance in the host. PR1 can competitively bind sterols, thereby inhibiting the growth of sterol-dependent pathogens and thus participating in antimicrobial defense. However, since sterols are rare in bacteria, PR1's regulation of plant antimicrobial function may depend on other domains or other mechanisms of action. Researchers have used phylogenetic mass spectrometry to analyze peptides induced in tomatoes after mechanical damage and methyl jasmonate treatment, identifying a polypeptide derived from the 11 C-terminal residues of the PR1 protein, named CAP-derived peptide 1 (CAPE1). It possesses a conserved PxGNxxxxxPY characteristic sequence, and the N-segment of the peptide cleavage site has a conserved CNYx motif, indicating that its processing endopeptidase is functionally conserved. Furthermore, CAPE1 may activate a novel damage-associated molecular pattern (DAMP) signaling pathway to induce the expression of plant defense response genes and immune activation. However, research on the function of CAPE1 has mainly focused on crops (such as tomatoes), while research on its function in woody plants is relatively scarce.

[0004] Identifying small peptides with disease-resistant functions in plants is an emerging and promising research field that helps develop alternative methods to improve plant disease resistance. However, the complex processing and maturation mechanisms, the difficulty in studying their biological functions, and the immature research techniques all contribute to the challenges of researching plant disease-resistant small peptides, but also provide greater opportunities. Applying plant disease-resistant small peptides to production practices is expected to reduce the use of pesticides and fertilizers, effectively protecting plant health and the ecological environment.

[0005] In view of this, the present invention selects the CAPE1 peptide fragments at the C-terminus of three PtPR1 proteins from Populus tomentosa to study their application in inducing a defensive response in poplar (or non-target plants such as chestnut) or improving the disease resistance of poplar or non-target plants. Summary of the Invention

[0006] In view of the technical problems existing in the background art, the purpose of the present invention is to provide the CAPE1 peptide segment at the C-terminus of PtPR1 protein and its application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides a CAPE1 peptide segment at the C-terminus of the PtPR1 protein, which is PtPR1f, PtPR1j, or PtPR1l, wherein the amino acid sequence of PtPR1f is shown in SEQ ID NO.1; the amino acid sequence of PtPR1j is shown in SEQ ID NO.2; and the amino acid sequence of PtPR1l is shown in SEQ ID NO.3.

[0009] A second aspect of the present invention provides the application of the CAPE1 peptide at the C-terminus of the above-mentioned PtPR1 protein in the prevention and control of diseases of woody plants.

[0010] Preferably, the woody plant is poplar or chestnut;

[0011] Preferably, the plant disease is caused by any one of the following fungi: Colletotrichum gloeosporioides, Populus canker, Botrytis cinerea, or Phytophthora spp.

[0012] Preferably, the method for preventing and controlling woody plant diseases using the CAPE1 peptide at the C-terminus of the PtPR1 protein includes the following steps: spraying or applying any one of the CAPE1 peptides at the C-terminus of the PtPR1 protein onto the surface of the woody plant.

[0013] The present invention has the following beneficial effects:

[0014] This invention provides three C-terminal CAPE1 peptides of the PtPR1 protein, namely PtPR1f, PtPR1j, or PtPR1l. Experimental results show that these three C-terminal CAPE1 peptides can induce immune defense responses in woody plants at low concentrations. The use of these three C-terminal CAPE1 peptides significantly improves the disease resistance of woody plants to *Colletotrichum gloeosporioides*, *Pseudomonas aeruginosa*, *Botrytis cinerea*, or *Phytophthora spp.*, without inhibiting the growth of these pathogens. Therefore, they can be applied to the prevention and control of woody plant diseases, providing a new approach to improving the disease resistance of woody plants and possessing broad application prospects. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a schematic diagram showing the classification results of the CAPE1 peptide segments at the C-terminus of the 12 PtPR1 proteins in Example 1;

[0017] Figure 2 The expression of the PtPR1 gene in poplar leaves after 0 h, 12 h, 24 h, and 36 h of infection by *Colletotrichum gloeosporioides*.

[0018] Figure 3 The results of treating poplar leaves with the CAPE1 peptide of three PtPR1 proteins, showing reactive oxygen species bursts and callose deposition.

[0019] Figure 4 Symptoms of disease after inoculation with *Colletotrichum gloeosporioides* following treatment of poplar leaves with CAPE1 peptides of three PtPR1 proteins.

[0020] Figure 5 Symptoms of poplar tree canker after inoculation with poplar canker pathogens following treatment of poplar branches with CAPE1 peptides of three PtPR1 proteins.

[0021] Figure 6 Symptoms of disease after inoculation with Staphylococcus aureus following treatment of poplar branches with CAPE1 peptides of three PtPR1 proteins.

[0022] Figure 7 Symptoms of disease after chestnut branches were treated with CAPE1 peptide fragments of three PtPR1 proteins and inoculated with chestnut blight pathogen.

[0023] Figure 8 The image shows the disease symptoms after treatment with CAPE1 peptides of three PtPR1 proteins 36 h after inoculating poplar branches with poplar rot pathogen.

[0024] Figure 9 The results showed that the CAPE1 peptide of the three PtPR1 proteins had no growth-inhibiting effect on *Colletotrichum gloeosporioides*, *Populus hygrophila*, *Botrytis cinerea*, and *Phytophthora spp.* Detailed Implementation

[0025] In the following description, specific details such as particular system architectures and techniques are set forth for illustrative purposes and not for limitation, in order to provide a thorough understanding of the embodiments of the invention. However, those skilled in the art will understand that the invention may be implemented in other embodiments without these specific details.

[0026] Example 1

[0027] 1. Screening

[0028] (1) Reference Figure 1 The CAPE1 peptides (PtPR1a-PtPR1l) of 12 PtPR1 proteins were divided into three categories: Category I, which have a conserved PxGNxxxxxPY motif and a conserved CNYx motif before the cleavage site; Category II, which have a conserved PxGNxxxxxPY motif but a non-conserved CNYx motif before the cleavage site; and Category III, which have a non-conserved PxGNxxxxxPY motif.

[0029] (2) Poplar leaves were infected with *Colletotrichum gloeosporioides* at 0 h, 12 h, 24 h, and 36 h, respectively. RNA was then extracted and samples were collected for detection of gene (PtPR1a-PtPR1l) expression levels. RNA extraction was performed using the TIANGEN RNA extraction kit according to the instructions, and the RNA content and quality were detected using Nanodrop.

[0030] (3) Reverse transcription to generate the first strand: 2 μg of RNA was used as a template, and cDNA was synthesized according to the instructions for use of the Hifair reverse transcriptase reagent from YEASEN. The volume was adjusted to 40 μL, and the reverse transcription product was diluted 4-fold. The expression level of poplar resistance-related genes was detected by real-time quantitative PCR. The primers used for the real-time quantitative PCR reaction are as follows:

[0031] PtActin upstream primer PtActin-5F:

[0032] 5'-TCATCGGAATGGAAGCTGCTGGTA-3';

[0033] PtActin downstream primer PtActin-3F:

[0034] 5'-TAGTGGAACCACCACTGAGCACAA-3';

[0035] PtPR1a upstream primer PtPR1a-5F

[0036] 5'-TTATGCTAACCAACGTGCCG-3';

[0037] PtPR1a downstream primer PtPR1a-3F:

[0038] 5'-AGCCTTCTCATCAACCCACA-3';

[0039] PtPR1b upstream primer PtPR1b-5F:

[0040] 5'-TTATGCTAACCAACGTGCCG-3';

[0041] PtPR1b downstream primer PtPR1b-3F:

[0042] 5'-AGCCATCTCATCAACCCACA-3';

[0043] PtPR1c upstream primer PtPR1c-5F:

[0044] 5'-ACAACACGGTAGCAGCCTAT-3';

[0045] PtPR1c downstream primer PtPR1c-3F:

[0046] 5'-CACCCACAAGTTCACTGCTG-3';

[0047] PtPR1d upstream primer PtPR1d-5F:

[0048] 5'-AATGTGGGCAAGTGAGAAGC-3';

[0049] PtPR1d downstream primer PtPR1d-3F:

[0050] 5'-GCCACACCCTAAATGAACCG-3';

[0051] PtPR1e upstream primer PtPR1e-5F:

[0052] 5'-ATGGGTCTCGGAGCGTAAAT-3';

[0053] PtPR1e downstream primer PtPR1e-3F:

[0054] 5'-TGAAAACTCCCCTTCCACGA-3';

[0055] PtPR1f upstream primer PtPR1f-5F:

[0056] 5'-CCTTGATGTGGGACTTTCAGC-3';

[0057] PtPR1f downstream primer PtPR1f-3F:

[0058] 5'-ACTTCTCTTCACCAGCCCAG-3';

[0059] PtPR1g upstream primer PtPR1g-5F:

[0060] 5'-TGCCCACAATAATGCTCGTG-3';

[0061] PtPR1g downstream primer PtPR1g-3F:

[0062] 5'-AACCCACAATTTCACCGCAG-3';

[0063] PtPR1h upstream primer PtPR1h-5F:

[0064] 5'-GACTTGTGCATTCTGGTGGG-3';

[0065] PtPR1h downstream primer PtPR1h-3F:

[0066] 5'-CCTGCATTCTCCACCAACAC-3';

[0067] PtPR1i upstream primer PtPR1i-5F:

[0068] 5'-TTGCAAACTTGAGCGACAGT-3';

[0069] PtPR1i downstream primer PtPR1i-3F:

[0070] 5'-ATTTCCTCCACACCACCTGT-3';

[0071] PtPR1j upstream primer PtPR1j-5F:

[0072] 5'-GAGATGCTACCCAATGCCAC-3';

[0073] PtPR1j downstream primer PtPR1j-3F:

[0074] 5'-CCCAGTGATCAACAGCCATG-3';

[0075] PtPR1k upstream primer PtPR1k-5F:

[0076] 5'-CCTTGTTTTCATGTGGGGCA-3';

[0077] PtPR1k downstream primer PtPR1k-3F:

[0078] 5'-CTCTCTCAAGGTTGTGGGGT-3';

[0079] PtPR1l upstream primer PtPR1l-5F:

[0080] 5'-ACTAAGGGACGGTTGCAGAA-3';

[0081] PtPR1l downstream primer PtPR1l-3F:

[0082] 5'-CCACCCACATTTTCACAGCA-3';

[0083] The PCR reaction system contained 0.5 μL cDNA, 10 μL qPCR SYBR Green Master Mix (Yeasen), 0.4 μL each of forward and reverse primers, and 8.7 μL water. The reaction program was: I: 95℃ for 2 min, II: 95℃ for 10 s, 60℃ for 30 s, with 40 cycles in step II. Data analysis was performed using 2... -ΔΔCT The method involved testing each sample three times, and the results are shown in [the table below]. Figure 2 .

[0084] Depend on Figure 2 The results showed that the expression levels of genes PtPR1a-PtPR1l were significantly increased. PtPR1f (amino acid sequence shown in SEQ ID NO.1), PtPR1j (amino acid sequence shown in SEQ ID NO.2), and PtPR1l (amino acid sequence shown in SEQ ID NO.3) were specifically selected from classes I, II, and III for further research.

[0085] 2. Study on the role of CAPE1 peptides (PtPR1f, PtPR1j, PtPR1l) at the C-terminus of three PtPR1 proteins in the control of woody plant diseases.

[0086] (1) Research on immune defense response

[0087] Reactive oxygen species (ROS) detection: Poplar leaves were immersed in the CAPE1 peptide segments (PtPR1f, PtPR1j, and PtPR1l) at the C-terminus of three selected PtPR1 proteins. After being vacuumed for 5-10 seconds, the leaves were allowed to stand for 15 minutes. Then, they were soaked in 3,3'-diaminobenzidine (DAB) solution (Solarbio) for 12 hours. The leaves were decolorized with 95% ethanol and observed. H2O was used as a negative control.

[0088] Callus deposition detection: Poplar leaves were treated with PtPR1f, PtPR1j, and PtPR1l according to method 1.1 above, and then allowed to stand for 15 h for complete decolorization. Afterward, the leaves were soaked in aniline blue for 12 h, and callus deposition was observed and detected using a fluorescence universal microscope (BX61). Each sample was repeated three times. Results are shown below. Figure 3 .

[0089] Depend on Figure 3 The results showed that the CAPE1 peptides at the C-terminus of the three PtPR1 proteins, namely PtPR1f, PtPR1j, and PtPR1l, could induce reactive oxygen species bursts and callosity deposition in poplar leaves.

[0090] (2) Study on the resistance of poplar leaves treated with CAPE1 peptide fragments (PtPR1f, PtPR1j, PtPR1l) at the C-terminus of three PtPR1 proteins to *Colletotrichum gloeosporioides*.

[0091] Using H2O as a control, PtPR1f, PtPR1j, and PtPR1l were sprayed or applied to poplar leaves for 24 hours. Five mm of *Colletotrichum gloeosporioides* mycelium was then inoculated onto the leaves, and the infection area was counted using Image J five days after inoculation. Each sample was tested three times. Results are shown in […]. Figure 4 .

[0092] Depend on Figure 4 The results showed that, compared with the negative control H2O treatment, the lesions infected with *Colletotrichum gloeosporioides* on poplar leaves treated with PtPR1f, PtPR1j, and PtPR1l were significantly reduced, and the resistance effect was PtPR1l>PtPR1j>PtPR1f. This result indicates that the CAPE1 peptide segments at the C-terminus of the three PtPR1 proteins provided in this invention, namely PtPR1f, PtPR1j, and PtPR1l, can significantly enhance the resistance of poplar leaves to *Colletotrichum gloeosporioides*.

[0093] (3) Study on the resistance of poplar branches treated with CAPE1 peptide fragments (PtPR1f, PtPR1j, PtPR1l) at the C-terminus of three PtPR1 proteins to poplar canker pathogen.

[0094] Poplar branches were disinfected with 75% alcohol and then scalded. The control group (CK) was used as a negative control, and the wild-type WT as a positive control. 50 μL of each of the three fungal agents (PtPR1f, PtPR1j, and PtPR1l) were applied to the wounds and allowed to absorb for 24 hours. Then, 5 mm of poplar rot fungal cake was inoculated into the wounds, and the cake was fixed in place with sealing film. The branches were placed in a well-ventilated, humid environment for 7 days, and the results were observed. The area of ​​lesions was statistically analyzed using ImageJ. Fifteen poplar branches were selected for each treatment. The results are shown below. Figure 5 .

[0095] Depend on Figure 5The results showed that, compared with the positive control WT treatment, the lesions infected by poplar canker fungus on poplar branches treated with the three C-terminal CAPE1 peptides of PtPR1 protein were significantly reduced, and the disease resistance effect was PtPR1l>PtPR1j>PtPR1f. These results indicate that the three C-terminal CAPE1 peptides of PtPR1 protein provided in this invention, namely PtPR1f, PtPR1j, and PtPR1l, can significantly enhance the disease resistance of poplar branches to poplar canker fungus.

[0096] (3) Study on the resistance of poplar branches treated with CAPE1 peptide fragments (PtPR1f, PtPR1j, PtPR1l) at the C-terminus of three PtPR1 proteins to Staphylococcus aureus.

[0097] Poplar branches were disinfected with 75% alcohol and then scalded. The control group (CK) was used as a negative control, and the wild-type WT as a positive control. 50 μL of each of the three fungal species (PtPR1f, PtPR1j, and PtPR1l) were applied to the wounds, and after 24 hours of absorption, 5 mm of *Staphylococcus aureus* mycelium was inoculated into the wounds. The inoculated mycelium was then fixed in place with sealing film. The branches were placed in a well-ventilated, humid environment for 10 days, and the results were observed. The area of ​​lesions was statistically analyzed using ImageJ. Fifteen branches were selected for each treatment. The results are shown below. Figure 6 .

[0098] Depend on Figure 6 The results showed that, compared with the positive control WT treatment, the number of lesions infected with *Botrytis cinerea* on poplar branches treated with PtPR1f, PtPR1j, and PtPR1l was significantly reduced, and the disease resistance effect was PtPR1l>PtPR1j>PtPR1f. This result indicates that the CAPE1 peptide segments at the C-terminus of the three PtPR1 proteins provided in this invention, namely PtPR1f, PtPR1j, and PtPR1l, can significantly enhance the disease resistance of chestnut branches to *Botrytis cinerea*.

[0099] (4) Study on the resistance of chestnut branches treated with CAPE1 peptides (PtPR1f, PtPR1j, and PtPR1l) at the C-terminus of three PtPR1 proteins to chestnut blight pathogen.

[0100] Chestnut branches were disinfected with 75% alcohol and then scalded. The control group (CK) was used as a negative control, and the wild-type WT group as a positive control. 50 μL of each of the three fungal agents (PtPR1f, PtPR1j, and PtPR1l) were applied to the wounds, and after 24 hours of absorption, 5 mm of *Pseudomonas aeruginosa* mycelium was inoculated into the wounds. The inoculated mycelium was then fixed in place with sealing film. The branches were placed in a well-ventilated, humid environment for 12 days, and the results were observed. The area of ​​lesions was statistically analyzed using ImageJ. Seven branches were selected for each treatment. The results are shown below. Figure 7 .

[0101] Depend on Figure 7 The results showed that, compared with the positive control WT treatment, the number of lesions infected by *P. chestnut blight* on chestnut branches treated with PtPR1f, PtPR1j, and PtPR1l was significantly reduced, and the disease resistance effect was PtPR1l>PtPR1j>PtPR1f. This result indicates that the CAPE1 peptide segments at the C-terminus of the three PtPR1 proteins provided in this invention, namely PtPR1f, PtPR1j, and PtPR1l, can enhance the disease resistance of chestnut branches to *P. chestnut blight*.

[0102] (5) Study on the spread of poplar rot pathogens on poplar branches

[0103] Poplar branches were disinfected with 75% alcohol and then scalded. The control group (CK) was used as a negative control, and the wild-type WT was used as a positive control. A 5 mm diameter seed cake of *Poplar rot* fungus was inoculated into the wound. After 36 hours, 50 μL of each of the three fungal agents (PtPR1f, PtPR1j, and PtPR1l) were applied to the wound, and the wounds were sealed with sealing film. The branches were placed in a well-ventilated, humid environment for 6 days, and the results were observed. The area of ​​lesions was statistically analyzed using ImageJ. Fifteen branches were selected for each treatment. The results are shown below. Figure 8 .

[0104] Depend on Figure 8 The results showed that, compared with the positive control WT treatment, the lesions of poplar branches treated with PtPR1f, PtPR1j, and PtPR1l were significantly reduced due to infection by poplar canker pathogens. This indicates that the CAPE1 peptide segments at the C-terminus of the three PtPR1 proteins provided in this invention, namely PtPR1f, PtPR1j, and PtPR1l, can significantly slow the spread of poplar canker pathogens on poplar branches.

[0105] (6) Study on the effects of the C-terminal CAPE1 peptides (PtPR1f, PtPR1j, PtPR1l) of three PtPR1 proteins on the growth of *Colletotrichum gloeosporioides*, *Poplar rot fungus*, *Botrytis cinerea*, and *Phytophthora spp.*

[0106] Activated *Botrytis cinerea*, *Poplar rot fungus*, *Colletotrichum gloeosporioides*, *Botrytis cinerea*, and *Phytophthora spp.* were inoculated onto PDA plates. When the bacteria had spread to 1 / 3 of the plate, two wells were punched on each side of the plate at a distance from the midpoint of the plate using a 5 mm punch. H2O was used as a control. 50 μL of each of the fungi (PtPR1f, PtPR1j, and PtPR1l) were added to the wells, and the plates were incubated at 25°C. Photos were taken when the bacteria reached the perimeter of the wells. Each sample was repeated three times. Results are shown below. Figure 9 .

[0107] Depend on Figure 9The results showed that, compared with the control H2O treatment, PtPR1f, PtPR1j, and PtPR1l did not inhibit the growth of *Colletotrichum gloeosporioides*, *Poplar rot fungus*, *Botrytis cinerea*, and *Chestnut blight fungus*.

[0108] This invention is not limited to the specific embodiments described above. Any modifications made by those skilled in the art based on the above concept without creative effort are within the scope of protection of this invention.

Claims

1. The application of the CAPE1 peptide at the C-terminus of the PtPR1 protein in the control of diseases in poplar or chestnut trees, wherein the CAPE1 peptide at the C-terminus of the PtPR1 protein is PtPR1f, and the amino acid sequence of PtPR1f is shown in SEQ ID NO.1, characterized in that... The disease is caused by any one of the following fungi: Colletotrichum gloeosporioides, Populus canker, Staphylococcus aureus, or Pseudomonas spp.

2. The CAPE1 peptide at the C-terminus of the PtPR1 protein, characterized in that, The CAPE1 peptide at the C-terminus of the PtPR1 protein is either PtPR1j or PtPR1l, wherein the amino acid sequence of PtPR1j is shown in SEQ ID NO.2; and the amino acid sequence of PtPR1l is shown in SEQ ID NO.

3.

3. The application of the CAPE1 peptide at the C-terminus of the PtPR1 protein as described in claim 2 in the prevention and control of diseases in poplar or chestnut trees, characterized in that... The disease is caused by any one of the following fungi: Colletotrichum gloeosporioides, Populus canker, Staphylococcus aureus, or Pseudomonas spp.

4. A method for controlling poplar or chestnut diseases using the CAPE1 peptide at the C-terminus of the PtPR1 protein, wherein the disease is caused by any one of *Colletotrichum gloeosporioides*, *Pseudomonas aeruginosa*, *Botrytis cinerea*, or *Phytophthora chestnutii*, and the CAPE1 peptide at the C-terminus of the PtPR1 protein is PtPR1f, PtPR1j, or PtPR1l, wherein... The amino acid sequence of PtPR1f is shown in SEQ ID NO.1; the amino acid sequence of PtPR1j is shown in SEQ ID NO.2; and the amino acid sequence of PtPR1l is shown in SEQ ID NO.

3. The method is characterized by the following steps: spraying or applying any one of the CAPE1 peptide segments at the C-terminus of the PtPR1 protein onto the surface of poplar or chestnut trees.

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