New use of colletotrichum gloeosporioides effector cfcpo1 mutants

Abstract

The application discloses a new use of a Guignardia citricarpa effector CfCPO1 mutant and relates to the technical field of molecular biology.The mutant of the Guignardia citricarpa effector CfCPO1 is applied to improving the resistance of Carya illinoensis, and the base sequence of the mutant is shown in SEQ ID NO.1 or SEQ ID NO.2.The pathogenicity of wild type, the CfCPO1 deletion mutant and the complementation strain is tested, and the results show that the CfCPO1 can degrade hydrogen peroxide in the Carya illinoensis, the Guignardia citricarpa carrying the CfCPO1 mutant is significantly weakened in the ability of decomposing H2O2, the H2O2 in the Carya illinoensis can be accumulated in time in the early stage of the Guignardia citricarpa infection, and the high expression of the defense-related genes of the Carya illinoensis is effectively activated, so that the Guignardia citricarpa infection is effectively hindered, and the resistance of the Carya illinoensis is improved.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and more specifically, relates to new applications of the effector CfCPO1 of *Discocephala spp.* and its mutants. Background Technology

[0002] Pecan (Carya illinoinensis (Wangenh) K. Koch), also known as thin-shelled pecan, is one of the most economically valuable nut species in the genus Carya of the Juglandaceae family. It originated in northern Mexico and the southern United States. Due to its extremely high nutritional value, pecan is widely cultivated globally. Large-scale cultivation in my country began around 2010, with major growing provinces including Anhui, Jiangsu, Zhejiang, and Yunnan. Although the national planting area reached approximately 85,000 hectares by 2022, the annual yield was only 4,500 metric tons. The main reason for this is that disease is one of the most critical factors limiting the yield and quality of pecans in China. Among the many fungi that infect pecans, several species of the genus *Colletotrichum* can cause anthracnose in thin-shelled pecans, including *Colletotrichum fructicola*, *Colletotrichum fioriniae*, *Colletotrichum siamense*, *Colletotrichum plurivorum*, and *Colletotrichum nymphaeae*. Of these, anthracnose caused by *Colletotrichum fructicola* is the most severe. One reason why anthracnose in thin-shelled pecans cannot be effectively controlled is that the pathogenic mechanism of *Colletotrichum fructicola* is still unclear.

[0003] In plant-pathogen interactions, effectors secreted by pathogens are the main determinants of pathogenicity. Effectors can enter host cells and target various immune-related proteins, thereby inhibiting the host's pathogen-associated molecular pattern (PAMP-Triggered Immunity, PTI) or effector-triggered immunological (ETI) responses, creating a favorable microenvironment for infection.

[0004] *Colletotrichum* fungi are listed as one of the ten most important genera of plant pathogenic fungi globally, and *Colletotrichum foetans* is one of the most important plant pathogens within this group. It can harm woody plants (including fruit trees and ornamental trees), vegetables, and field crops. However, research on *Colletotrichum foetans* effectors is still in its early stages. It is noteworthy that existing studies have shown that the functions of *Colletotrichum foetans* effectors exhibit significant host dependence. For example, the effector *CfXyn11A* triggers a reactive oxygen species burst and programmed cell death (immune response) in *Nicotiana benthamiana*, but in its natural host, pear, it evades recognition and instead functions to degrade cell walls and acquire nutrients. This finding suggests that even the same effector may have fundamentally different functions and modes of action in different host contexts. Therefore, directly extrapolating the conclusions of existing effector studies on apples, camellia oleifera, or pears to pecans lacks scientific basis.

[0005] Specifically, the following key gaps exist in the existing research: (1) Regarding effectors that induce plant cell necrosis, Chen et al. and Hu et al. identified 4 and 2 candidate effectors respectively from the early transcriptome of *Colletotrichum oryzae* infecting Camellia oleifera and pecan, but their functions have not been further studied; (2) Regarding effectors that inhibit plant immunity, effector CfEC28 inhibits immunity by manipulating DAHPS-mediated metabolic flux in apple chloroplasts; effector CfEC12 interacts with apple MdNIMIN2 to interfere with the salicylic acid pathway; effector CfEC92 inhibits BAX-triggered necrosis in *Nicotiana benthamiana*, and its toxicity to Camellia oleifera is significantly reduced after knockout; and effector CfCFEM21 inhibits BAX / INF1-induced programmed cell death in *Nicotiana benthamiana*; the sporulation of the CfE15 deletion mutant is reduced, resulting in a significant decrease in the pathogenicity of *Colletotrichum oryzae* to strawberry, but none of these studies involve pecan. Existing research has not yet identified any key effector secreted in the interaction between *Colletotrichum spp.* and pecans; (3) As a perennial woody plant of the Juglandaceae family, pecans have a long fruit development period and abundant secondary metabolites, and their immune system may have unique composition and regulatory characteristics, which are significantly different from those of plants of the Rosaceae and Theaceae families. Therefore, a large number of *Colletotrichum spp.* effectors still need to be identified, and their functions in the process of *Colletotrichum spp.* infecting pecans urgently need to be elucidated. Summary of the Invention

[0006] To address the aforementioned problems in the existing technology, the technical problem to be solved by this invention is to provide the application of a mutant of the fruit-borne scab effector CfCPO1 in improving plant resistance. Another technical problem to be solved by this invention is to provide the application of the fruit-borne scab effector CfCPO1 in the degradation of hydrogen peroxide.

[0007] To solve the above-mentioned technical problems, the technical solution adopted in this application is as follows:

[0008] Application of mutants of the fruit-borne thorn spore effector CfCPO1 in improving the resistance of thin-shelled pecans.

[0009] The application described herein, wherein the mutant base sequence is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0010] The application steps are as follows:

[0011] 1) Amplify the upstream and downstream flanking sequences of the CfCPO1 gene using primer pairs CfCPO1-up F / R and CfCPO1-down F / R, respectively; ligate the two PCR products to both sides of the hygromycin B phosphotransferase expression cassette, and construct the gene replacement fragment by overlapping PCR using primer pair CfCPO1-up F / CfCPO1-down R;

[0012] 2) The purified gene replacement fragment was transformed into the protoplasts of the wild-type strain CZ102;

[0013] 3) Candidate transformants were screened on TB3 plates and verified by PCR using primer pairs CfCPO1-inner F / R and CfCPO1-outer F / R, respectively; finally, two CfCPO1 knockout mutants, KO-1 and KO-4, were obtained. The sequence of CfCPO1 knockout mutant KO-1 is shown in SEQ ID NO.1, and the sequence of CfCPO1 knockout mutant KO-4 is shown in SEQ ID NO.1.

[0014] The primer sequences for the aforementioned application are as follows:

[0015] CfCPO1-up F:5'-CCGGTCAACTTGCAATATCTC-3',

[0016] CfCPO1-up R: 5'-TTGACCTCCACTAGCTCCAGCCAAGCCGAATGTTGGAAATGCGTAGTTG-3';

[0017] CfCPO1-down F: 5'-CAAAGGAATAGAGTAGATGCCGACCGATGTGCTTCTGCTTCGGTGTA-3',

[0018] CfCPO1-down R: 5'-CTTAACTCCGGCAAGTCAAGT-3';

[0019] CfCPO1-inner F: 5'-GCCTTGGACGCCAACAGATC-3',

[0020] CfCPO1-inner R: 5'-GACGTTCATGTACTTAGTCGG-3';

[0021] CfCPO1-outer F: 5'-GTTGGTGTCTACAAGTTACAAC-3',

[0022] CfCPO1-outer R: 5'-CTTTGTTTGCGGCGCCCTTGA-3'.

[0023] Application of mutants of the fruit-borne thorn spore effector CfCPO1 in activating high expression of defense-related genes in thin-shelled pecans; the base sequence of the mutant is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0024] The application in question involves CiPRO-10 and CiPTI6 as the relevant genes.

[0025] Application of the effector CfCPO1 of the fruit-borne thorn cluster spore in the degradation of hydrogen peroxide in thin-shelled pecans

[0026] Beneficial effects: Compared with the prior art, the technical advantages of this invention are:

[0027] 1) This application screened a candidate effector CfCPO1 from genes upregulated in the early stage of pecan infection by *Colletotrichum spp.*, and determined its conservation in *Colletotrichum* species through phylogenetic analysis. Furthermore, an Agrobacterium-mediated transient expression experiment confirmed that the effector CfCPO1 can inhibit cell death triggered by the pathogen-associated molecular pattern INF1.

[0028] 2) This application tested the pathogenicity of wild-type, CfCPO1 deletion mutant and complement strain. The results showed that CfCPO1 can degrade hydrogen peroxide in pecans. The fruit-bearing Colchicum carrying the CfCPO1 mutant had a significantly weakened ability to decompose H2O2, which led to the timely accumulation of H2O2 in the early stage of Colchicum infection in pecans. It also effectively activated the high expression of defense-related genes in pecans, thereby effectively inhibiting the infection of Colchicum and improving the resistance of pecans. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the CfCPO1 structural domain;

[0030] Figure 2The results of CfCPO1 inhibiting programmed cell death are shown in the figure. Among them, a) is a representative leaf of Nicotiana benthamiana 7 days after injection of Agrobacterium carrying the target gene, b) cell death of representative Nicotiana benthamiana leaves under ultraviolet light, c) ion permeation determination of the degree of cell death in Nicotiana benthamiana, and d) immunoblotting analysis of the target protein.

[0031] Figure 3 This image shows the expression pattern of CfCPO1 in the early stage of *Colletotrichum crenulata* infection and the results of its infection-promoting function. A) shows the expression pattern of CfCPO1 in the early stage of infection; b) shows that CfCPO1 can promote the infection of *Colletotrichum crenulata* on *Nicotiana benthamiana*. Left side: *Nicotiana benthamiana* leaves were first injected with pBINGFP-CfCPO1, and 36 hours later, 5 mm mycelial blocks of *Colletotrichum crenulata* were inoculated. Right side: Control. Lesions were observed under UV light 48 hours after inoculation.

[0032] Figure 4 This is a diagram showing the construction results of the CfCPO1 knockout mutant and the complementary mutant. In the diagram, the internal and external primers ab confirm that CfCPO1 was successfully replaced, and the mycelial fluorescence observation and protein blot analysis of cd confirm that the complemented strains KO-1-C and KO-4-C were successfully obtained.

[0033] Figure 5 The figure shows the results of CfCPO1 participating in the degradation process of H2O2; in the figure, a) wild type (WT), knockout mutants KO-1 and KO-4 and the reintroduced strain KO-1-C were cultured on PDA medium supplemented with 0.06 mol / L H2O2 for 4 days; b) statistical analysis of the colony diameter of the tested strains.

[0034] Figure 6 This is a graph showing the contribution of CfCPO1 to the pathogenicity of *Colletotrichum spp.* In the graph, a) is the pathogenicity determination of wild-type (WT), knockout mutant KO-1, and complement strain KO-1-C; b) is the statistical analysis of lesion diameters on leaves 5 days after inoculation with wild-type, knockout mutant KO-1, and complement strain KO-1-C; c) is the hydrogen peroxide content in pecans 3, 6, 12, and 24 hours after inoculation with wild-type and knockout mutant KO-1; and d) is the relative expression levels of defense-related genes in pecans 3 days after inoculation with wild-type, knockout mutant KO-1, and complement strain KO-1-C. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art.

[0036] Example 1: Bioinformatics and Systemic Evolutionary Analysis of CfCPO1

[0037] Based on the transcriptome annotation information of *Colletotrichum gloeosporioides*, a gene with gene ID CGMCC3_g4564, named CfCPO1, and a sequence length of 741 bp was selected for bioinformatics analysis. The results showed that the CfCPO1 cDNA encodes a 246-amino acid sequence containing a 19-aa signal peptide at its N-terminus, but lacking any transmembrane domain. Furthermore, this protein contains a domain (12-244aa)... Figure 1 ).

[0038] Phylogenetic analysis of CfCPO1 was performed, and based on the phylogenetic distribution of CfCPO1 homologous proteins in different organisms, a total of 44 genes encoding homologous protein sequences were identified. Among them, 37 genes belong to the genus *Colletotrichum* and 7 belong to the genus *Diaporthe*. The results indicate that CPO1 is ubiquitous in the genus *Colletotrichum*.

[0039] Example 2: CfCPO1 Inhibition of PAMP-Triggered Cell Necrosis Detection

[0040] The CfCPO1 and CfCPO1-SP genes (gene ID: CGMCC3_g4564) were cloned from the cDNA of *Colletotrichum cirrhosa* using specific primers. The purified PCR products were ligated into the pBINGFP vector using ApexHF HS DNA Polymerase FS (Aikerui Biotechnology Co., Ltd., Changsha, Hunan). The empty vector pBINGFP served as a control. Primer sequences are detailed in Table 1.

[0041] Table 1 Primers used for amplifying the target fragment

[0042]

[0043]

[0044] The constructed CfCPO1, CfCPO1-SP, and empty vector pBINGFP plasmids were inserted into Agrobacterium tumefaciens GV3101 cells via electroporation. GV3101 cells carrying the constructed plasmids were washed with washing buffer, and the OD... 600Adjust the concentration to 0.5. Agrobacterium cells carrying the corresponding plasmid were infiltrated into tobacco leaves. Sixteen hours later, Agrobacterium cells carrying the pathogen-associated molecular pattern (PAMP) INF1 of Phytophthora infestans were injected again at the same injection site. Five to seven days after infiltration injection, the results showed that CfCPO1 could inhibit INF1-induced cell necrosis, while CfCPO1-SP and the negative control pBINGFP could not inhibit INF1-induced cell necrosis. Figure 2 a).

[0045] The degree of cell necrosis in *Agrobacterium benzoate* leaves was quantified by detecting ion permeability. The specific method was as follows: Holes were punched at the injection sites using a 9 mm diameter punch, with five replicates for each treatment. Five leaflets from the same treatment were collected and suspended in 5 mL of ddH₂O, then soaked at room temperature for 3 h. The conductivity was measured using a conductivity meter, and the value was recorded as value A. After reading the value, 5 mL of the suspension was boiled in boiling water for 30 min. Once the suspension cooled to room temperature, its conductivity was measured again, and the value was recorded as value B. Finally, the degree of cell necrosis caused by the transiently expressed proteins of *Agrobacterium benzoate* was calculated using the formula: ion permeability = (A / B) × 100%.

[0046] The ion permeability results further confirmed that the cell necrosis rate induced by CfCPO1 injection followed by INF1 injection was significantly lower than that induced by CfCPO1-SP and empty vector pBINGFP injection followed by INF1 injection. Figure 2 b).

[0047] To further confirm whether the tested protein was expressed, protein extraction and Western blot analysis were subsequently performed. The specific steps for protein extraction were as follows: NP-40 lysis buffer (Beyotime, P0013F) was taken, and the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) was added to bring the final PMSF concentration to 1 mM. Leaf samples of *Nicotiana benthamiana* injected with *Agrobacterium* for 36–48 h were collected, ground into powder using liquid nitrogen, and the powder was added to the lysis buffer. The mixture was then vortexed for 3 min, centrifuged at 13000 r and 4 ℃ for 10 min, and the supernatant was transferred to a new centrifuge tube for further centrifugation. After centrifugation at 13000 r and 4 ℃ for 5 min, the supernatant was transferred to a new centrifuge tube and stored at -80 ℃ for later use.

[0048] The specific procedure for Western blot is as follows: 1) Mix the protein with 5 Mix loading buffer (10 μL loading buffer + 40 μL protein) thoroughly, then incubate in boiling water for 5 minutes to denature. 2) Assemble the protein gel apparatus and add 1 Running buffer. Then add 10-20 μL of protein and 7 μL of protein marker to the wells of the protein gel for SDS-PAGE electrophoresis. First, run the electrophoresis at 80 V. After the sample reaches the lower protein layer, switch to 120 V. 3) Prepare polyvinylidene fluoride (PVDF) membrane, filter paper, sponge, protein gel, and clamps. Place them in the following order: black side of clamp (negative electrode), sponge pad, filter paper, protein gel, PVDF membrane, filter paper, sponge pad, and white side of clamp (positive electrode). Add transfer buffer to the electrophoresis tank. Run at 80 V on ice for 2.5 h (soak the PVDF membrane in methanol for 15 s before use). 4) Add 20 mL of PBST containing 4% skim milk powder and block on a shaker at 50 r for 1 h. 5) Discard the blocking solution and add 3 6) After recovering the primary antibody, wash the membrane with PBST containing 4% skim milk powder at a ratio of 1:5000. Incubate on a shaker at 50 rpm for 1.5 h. 7) Add PBST containing 4% skim milk powder again, then add the fluorescent secondary antibody at a ratio of 1:10000. Incubate on a shaker at 50 rpm for 0.5 h. 8) After recovering the secondary antibody, wash the membrane three times with PBST in the dark, each time at 50 rpm for 5 min. After washing, add PBST again and store at 4 ℃ or scan the membrane directly. 9) Perform scanning imaging using an infrared fluorescence scanning imager. 10) Ponceau S staining: After soaking the membrane in Ponceau S staining solution for 5 minutes, wash it off with 10% acetic acid, wash off the residual staining solution, and then take a picture.

[0049] Western blot analysis showed that all tested proteins were expressed normally. Figure 2 c).

[0050] Example 3: Expression pattern and infection-promoting ability of CfCPO1 during the infection stage of *Colletotrichum flavomarginata*

[0051] Cultivate the conidia of *Colletotrichum candida* in a complete culture medium by shaking, and adjust the conidia concentration to 1×10⁻⁶. 5The spore suspension was sprayed onto the leaves of 3-month-old pecan seedlings. Samples were collected at 3, 6, 12, and 24 hours post-inoculation, and total RNA was extracted. The total RNA was then reverse transcribed into cDNA using a reverse transcription kit for subsequent qRT-PCR detection.

[0052] qRT-PCR results showed that the expression level of CfCPO1 significantly increased at 3, 6, 12 and 24 h post-inoculation, reaching a peak at 3 h. Figure 3 a). This indicates that CfCPO1 is involved in the early infection process and can affect the virulence of *Colletotrichum oryzae*.

[0053] Agrobacterium carrying CfCPO1 and the empty vector pBINGFP were transiently expressed in Nicotiana benthamiana for 36-48 hours, and then inoculated with *Colletotrichum citrinum*. Two days after inoculation, the size of lesions caused by *Phytophthora capsici* was observed. The results showed that the lesion area of ​​Nicotiana benthamiana leaves pre-injected with pBINGFP-CfCPO1 before inoculation with *Colletotrichum citrinum* was significantly larger than that of leaves pre-injected with pBINGFP (empty vector control). Figure 3 b). This indicates that CfCPO1 can promote the infection of *Discocephala spp.* on *Nicotiana benthamiana*.

[0054] Example 4: Detection of the ability of CfCPO1 to degrade hydrogen peroxide

[0055] The gene was knocked out using a homologous recombination substitution strategy. The specific method is as follows:

[0056] First, the upstream and downstream flanking sequences (approximately 1500 bp) of the CfCPO1 gene were amplified using primer pairs CfCPO1-up F / R and CfCPO1-down F / R, respectively. The two PCR products were ligated to both sides of a hygromycin B phosphotransferase (HPH) expression cassette, and a gene substitution fragment was constructed using overlap PCR and primer pair CfCPO1-up F / CfCPO1-down R. Second, the purified gene substitution fragment was transformed into protoplasts of wild-type strain CZ102. Third, candidate transformants were screened on TB3 plates and verified by PCR using primer pairs CfCPO1-inner F / R and CfCPO1-outer F / R, respectively. Two CfCPO1 knockout mutants, KO-1 and KO-4, were finally obtained. The homologous substitution sequence of the CfCPO1 gene in knockout mutant KO-1 is shown in SEQ ID NO.1, and the homologous substitution sequence of the CfCPO1 gene in knockout mutant KO-4 is shown in SEQ ID NO.2. Further PCR verification using inner and outer primers (primers shown in Table 1) confirmed that CfCPO1 had been successfully replaced. Figure 4 ab).

[0057] Subsequently, the replenishment vector TOP161-CfCPO1 was constructed. The specific method is as follows:

[0058] The coding region (CDS) and GFP gene of CfCPO1 were amplified using primer pairs TOP161-CfCPO1 F / R and TOP161-GFP F / R, respectively. The two PCR products were ligated and inserted into the linearized vector TOP161. The resulting fusion construct TOP161-CfCPO1-GFP was verified by sequencing and then transformed into protoplasts of a CfCPO1 knockout mutant. Further mycelial fluorescence observation and Western blot analysis showed that the complement strains KO-1-C and KO-4-C were also successfully constructed. Figure 4 cd).

[0059] In this example, after adding 0.06 mol / L H2O2 to PDA plates, wild-type, knockout mutants KO-1 and KO-4, and the complement strain KO-1-C were inoculated, and their growth was observed. The results showed that compared to the wild type, the knockout mutants KO-1 and KO-4 grew more slowly on PDA medium containing 0.06 mol / L H2O2. However, the complement strain KO-1-C recovered its resistance to 0.06 mol / L H2O2, and its colony size was similar to that of the wild type. Figure 5 a). Furthermore, the colony diameters of KO-1 and KO-4 were significantly smaller than those of the wild type and KO-1-C, while there was no significant difference between the wild type and KO-1-C. Figure 5 b). This indicates that CfCPO1 participates in the degradation process of hydrogen peroxide.

[0060] Example 5: Effect of CfCPO1 on the pathogenicity of *Colletotrichum cirrhosa*

[0061] This embodiment tests the pathogenicity of wild-type, CfCPO1 deletion mutant, and complemented strains. The conidial suspensions of wild-type, CfCPO1 deletion mutant, and complementary strains were adjusted to 1×10⁻⁶. 5 1 spores / mL. 15 μL of conidial suspension from each strain was inoculated onto healthy pecan leaves (three-month-old thin-shelled pecan seedlings). The inoculated seedlings were placed in a 25℃ humidified incubator. Disease symptoms were photographed and the size of corresponding lesions was measured 5 days after inoculation.

[0062] Leaves of *Caryophyllum pekinensis* were collected 3 hours after inoculation with wild-type, CfCPO1 deletion mutant, and complement strains, respectively. After quick-freezing in liquid nitrogen, RNA was extracted, and the expression levels of defense-related genes in *Caryophyllum pekinensis* were subsequently detected. Simultaneously, leaf tissues of *Caryophyllum pekinensis* were collected at 3, 6, 12, 24, and 48 hours after inoculation with wild-type and CfCPO1 deletion mutants, respectively, to detect the H2O2 content in the leaves.

[0063] The results showed that the lesions produced by the knockout mutants KO-1 and KO-4 were significantly smaller than those produced by the wild-type and complement strain KO-1-C. Figure 6 ab). The H2O2 content in pecans inoculated with the wild-type strain was lowest at 3 h after inoculation, then significantly increased at 6 h and 12 h, and subsequently decreased significantly at 24 h. Conversely, the H2O2 content in pecans inoculated with the knockout mutant KO-1 strain was also lowest at 3 h after inoculation, and then gradually increased. Notably, the H2O2 content in pecans inoculated with the wild-type strain was significantly lower than that in pecans inoculated with the knockout mutant KO-1 strain at 3 h, 6 h, and 24 h. Figure 6 c). Simultaneously, 3 hours post-inoculation, compared to inoculation with wild-type and complement strain KO-1-C, the expression levels of defense-related genes CiPRO-10 (gene ID: CiPaw.01G143200.1) and CiPTI6 (gene ID: CiPaw.14G049500.1) were significantly increased in thin-shelled pecans inoculated with the knockout mutant KO-1. Figure 6 d).

[0064] The above results indicate that CfCPO1 can degrade hydrogen peroxide in pecans. The CfCPO1 mutant of *Colletotrichum fusiforme* has a significantly weakened ability to decompose H2O2, which leads to the timely accumulation of H2O2 in the early stage of *Colletotrichum fusiforme* infection. Furthermore, it effectively activates the high expression of defense-related genes in pecans, thereby effectively inhibiting the infection of *Colletotrichum fusiforme* and improving the resistance of pecans.

[0065] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. Application of mutants of the fruit-borne thorn spore effector CfCPO1 in improving the resistance of thin-shelled pecans.

2. The application according to claim 1, wherein the mutant base sequence is as shown in SEQ ID NO.1 or SEQ ID NO.

2.

3. The application according to claim 1, characterized in that, The steps are as follows: 1) Amplify the upstream and downstream flanking sequences of the CfCPO1 gene using primer pairs CfCPO1-up F / R and CfCPO1-down F / R, respectively; ligate the two PCR products to both sides of the hygromycin B phosphotransferase expression cassette, and construct the gene replacement fragment by overlapping PCR using primer pair CfCPO1-up F / CfCPO1-down R; 2) The purified gene replacement fragment was transformed into the protoplasts of the wild-type strain CZ102; 3) Candidate transformants were screened on TB3 plates and verified by PCR using primer pairs CfCPO1-inner F / R and CfCPO1-outer F / R, respectively; finally, two CfCPO1 knockout mutants, KO-1 and KO-4, were obtained. The sequence of CfCPO1 knockout mutant KO-1 is shown in SEQ ID NO.1, and the sequence of CfCPO1 knockout mutant KO-4 is shown in SEQ ID NO.

1.

4. The application according to claim 3, characterized in that, The primer sequences are as follows: CfCPO1-up F:5'-CCGGTCAACTTGCAATATCTC-3', CfCPO1-up R: 5'-TTGACCTCCACTAGCTCCAGCCAAGCCGAATGTTGGAAATGCGTAGTTG-3'; CfCPO1-down F: 5'-CAAAGGAATAGAGTAGATGCCGACCGATGTGCTTCTGCTTCGGTGTA-3', CfCPO1-down R: 5'-CTTAACTCCGGCAAGTCAAGT-3'; CfCPO1-inner F: 5'-GCCTTGGACGCCAACAGATC-3', CfCPO1-inner R: 5'-GACGTTCATGTACTTAGTCGG-3'; CfCPO1-outer F: 5'-GTTGGTGTCTACAAGTTACAAC-3', CfCPO1-outer R: 5'-CTTTGTTTGCGGCGCCCTTGA-3'.

5. Application of mutants of the fruit-borne thorn spore effector CfCPO1 in activating high expression of defense-related genes in thin-shelled pecans; the base sequence of the mutant is shown in SEQ ID NO.1 or SEQ ID NO.

2.

6. The application according to claim 5, characterized in that, The relevant genes mentioned are CiPRO-10 and CiPTI6.

7. Application of the fruit-borne thorn spore effector CfCPO1 in the degradation of hydrogen peroxide in thin-shelled pecans.

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