Application of LtSCRE11 protein in regulation and control of resistance of peach trees to gummosis

Abstract

The invention relates to an application of LtSCRE11 protein in regulating and controlling resistance of peach trees to gummosis. Micromolecular cysteine-rich protein LtSCRE11 secreted by peach gummosis pathogenic bacteria, namely, dispora pirocolor, is screened by a method of combining a genome and a plant bacterium interaction transcriptome with bioinformatics. It is found through injection of nicotiana benthamiana that LtSCRE11 can induce ROS bursting and callus accumulation, and injection of purified LtSCRE11 protein into nicotiana benthamiana and peaches can induce expression of plant immune related genes and MAPK protein phosphorylation signals, so that resistance of nicotiana benthamiana and peaches to pathogenic bacteria is induced. The invention proves that the LtSCRE11 plays a positive role in coping with the infection of pathogenic bacteria by plants, the resistance of peaches to the pathogenic bacteria of the gummosis can be remarkably improved by injecting the LtSCRE11 protein into leaves of peach trees, and the LtSCRE11 protein is expected to be developed into a novel immune resistance inducer target for preventing and controlling the gummosis of the peach trees.

Description

Technical Field

[0001] The present invention relates to the field of methods for improving the gummosis resistance of peach trees, and particularly to an application of a LtSCRE11 protein in regulating the gummosis resistance of peach trees. Background Art

[0002] Peach gummosis is a common fruit tree disease in my country's peach-producing regions, with particular severity in the Yangtze River Basin and areas south of it. This disease causes necrosis of peach tree branches and trunks, accompanied by extensive colloid exudation. This leads to nutrient loss, branch dieback, and ultimately shortened tree lifespan, severely impacting peach yield and quality. Various fungi have been reported to cause peach gummosis, with Botryosphaeria being the primary cause. Among these, Lasiodiplodia theobromae JMB122 has demonstrated strong pathogenicity.

[0003] Plant innate immunity includes PTI (Pattern-triggered immunity) and ETI (Effector-triggered immunity). The PTI response is the primary battlefield for plants to respond to foreign microbial invasion. Pattern recognition receptors (PRRs) located on the cell membrane recognize pathogen-associated molecular patterns (PAMPs) from microorganisms, thereby activating a series of immune responses, such as ROS (reactive oxygen species) bursts, callose accumulation, and MAPK protein phosphorylation cascade immune signaling. However, there are no reports on the recognition of PAMPs and the activation of immune responses in peach. Therefore, exploring the mechanism by which receptors recognize PAMPs and activate immune responses during gummosis infection is of great theoretical significance for the precise prevention and control of peach gummosis.

[0004] Experimental content and technical methods:

[0005] Using genomic and transcriptomic approaches combined with bioinformatics, we identified LtSCRE11, a protein secreted by the peach gummosis pathogen L. theobromae. Injection of LtSCRE11 into Nicotiana benthamiana plants revealed that it induced ROS production and callose accumulation. Furthermore, injection of purified LtSCRE11 protein into Nicotiana benthamiana and peach plants induced the expression of plant immunity-related genes and MAPK phosphorylation, thereby inducing resistance to the pathogen in both plants. This confirms that LtSCRE11 is a PAMP molecule that plays an active role in plant response to pathogen infection and can enhance plant resistance to pathogens. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to address the above problems and requirements and provide an application of LtSCRE11 protein in regulating the resistance of peach trees to gummosis.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] Use of LtSCRE11 protein in regulating resistance to peach gummosis, wherein the LtSCRE11 protein is selected from any one of the following (a) or (b):

[0009] (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0010] (b) A protein derived from (a) having the same enzymatic activity as that in (a) wherein the amino acid sequence is substituted, deleted or added with one or more amino acid sequences.

[0011] Furthermore, the specific application method is: using LtSCRE11 protein injection to treat peach tree leaves.

[0012] Furthermore, the LtSCRE11 protein is a purified LtSCRE11 protein diluted to a concentration of no more than 5 μM.

[0013] The beneficial effects of the present invention are:

[0014] Using a genomic and transcriptomic approach combined with bioinformatics, we identified LtSCRE11, a protein secreted by the bacterium Lactodiplodia theobromae. Injection of LtSCRE11 into Nicotiana benthamiana plants revealed that it can induce ROS production and callose accumulation. Furthermore, injection of purified LtSCRE11 protein into Nicotiana benthamiana and peach plants induced the expression of plant immunity-related genes and MAPK phosphorylation, thereby inducing pathogen resistance in both plants. This confirms that LtSCRE11 is a PAMP molecule that plays an active role in plant response to pathogen infection and can enhance peach resistance to gummogenic pathogens.

[0015] The present invention is described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the phylogenetic tree of LtSCRE11 and its homologous proteins in different pathogens;

[0017] Figure 2 LtSCRE11 induces ROS burst and callose accumulation;

[0018] Figure 3is the relative expression level of immune-related genes in peach and Nicotiana benthamiana after induction by LtSCRE11;

[0019] Figure 4 The activation of MAPK phosphorylation signaling after injection of LtSCRE11 protein into peach and Nicotiana benthamiana;

[0020] Figure 5 The lesion area and relative biomass of peach and Nicotiana benthamiana plants after injection of LtSCRE11 protein and inoculation with fungus nematode and Phytophthora capsici, respectively. DETAILED DESCRIPTION

[0021] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0022] 1. Bioinformatics Analysis of LtSCRE11

[0023] The CDS of LtSCRE11 is 435 bp long and encodes 144 amino acids. We used the amino acid sequence of LtSCRE11 to search for homologous sequences retrieved from the NCBI database and constructed a phylogenetic tree of homologous proteins from a series of different pathogens, including 21 fungi and 11 oomycetes. The results showed that ( Figure 1 LtSCRE11 homologs are widely found in fungi and oomycetes, particularly common crop pathogens such as Phytophthora sojae, P. infestans, P. capsici, and Fusarium oxysporum. A phylogenetic tree was constructed using IQ-TREE (with a bootstrap value set to 1000) based on maximum likelihood and visualized using iTOL.

[0024] The CDS sequence of the LtSCRE11 gene is as follows:

[0025] ATGCGCTTCACCACCTTCGCCGCTGTGCTCGCAACCGCCGCCACCGTGTCGGCGACGCTCGACCCGTGCACGTCCAACACCAAGGGCAAGGTGCCCAAGAGCCCGGCGTGCTCGGCGACCAAGAAGTCGACCAACATTCAGGCCGCTGAGTGCTCGCACAACACGCGTGTCTCCGGCACCCAGACCTTCGCTGTCTGGACTCGCACCAAGACCAACTCCAACGGCATCTCGTACGGCACCTGCGAGGCCTACACCTGCACCGCCCCCACCGATAGCCAGCTCACCGCCAACTCCGACGGCTGGACCTTCTTCTGGAATGACAACGGCGAGTCCTCTGGCGTCGGCACTGGTTGCATCAGGGACCCCAACACCACCGAGTGCGGTTGCGAGAACTCGGACGGCGAGTTCATCGTCGGCAGCGACTCTTGCAAATAG(SEQ ID NO:2)

[0026] The amino acid sequence of the LtSCRE11 protein is as follows:

[0027] MRFTTFAAVLATAATVSATLDPCTSNTKGKVPKSPACSATKKSTNIQAAECSHNTRVSGTQTFAVWTRTKTNSNGISYGTCEAYTCTAPTDSQLTANSDGWTFFWNDNGESSGVGTGCIRDPNTTECGCENSDGEFIVGSDSCK(SEQ ID NO:1)

[0028] 2. Induction and purification of the LtSCRE11 protein

[0029] Using the one-step cloning technology, the pET-32a (containing a His tag) vector plasmid was first enzyme-digested and linearized (the enzyme cutting sites were EcoRⅠ and XhoⅠ); homology arm primers were designed for the full-length coding sequence of the LtSCRE11 gene, and the target fragment was ligated to the pET-32a vector using the ClonExpress II One Step Cloning Kit (Vazyme, China). The above ligation products were transformed into DH5α competent cells. The vector resistance was Amp resistance. The successfully constructed recombinant plasmid pET-32a-LtSCRE11 was stored at -20°C for future use. The empty vector pET-32a was used as a control. The above plasmid was then transformed into BL21 (DE3) competent cells and spread on LB plates. Positive single clones were picked and cultured overnight. Subsequently, the overnight culture solution was taken at a ratio of 1:100 and inoculated into 1 L liquid LB medium containing the corresponding antibiotics. The culture was cultured at 37°C and 200 rpm for 2 h until the OD 600 The concentration of IPTG was 0.6. When the bacterial solution was allowed to stand and cool to 16°C, IPTG was added to a final concentration of 0.5mM. The culture was continued at 16°C and 120rpm for 24h. The bacterial solution was collected into a centrifuge tube and centrifuged at 4°C and 12000rpm for 1min. The supernatant was discarded and the precipitate was collected. 80mL of non-denaturing lysis buffer was added to fully resuspend the bacteria. PMSF was added to the lysate to a final concentration of 1mM and lysozyme was added to the lysate to a final concentration of 1mg / mL. After mixing, the suspension was allowed to stand on ice for 30min. The suspended bacteria were crushed using an ultrasonic disruptor on ice (ultrasonic power 300W, ultrasonic treatment for 10s each time, 10s interval each time, a total of 6 ultrasonic treatments, alarm temperature 4°C), the lysate was observed at any time, and ultrasonication was stopped immediately when it was clear. Centrifuged at 4°C and 12000rpm for 30min, the supernatant of the bacterial lysate was collected and placed on ice: refer to BeyoGold TM The supernatant protein was purified according to the instructions of His-tag Purification Resin (Beyotime, China); desalting and concentration were performed using ultrafiltration centrifuge tubes with a molecular weight cutoff of 10 kDa (Millipore, USA); the specific method was referred to the instructions; finally, the protein concentration was measured, and the protein was aliquoted and quickly frozen in liquid nitrogen and stored in a -80°C freezer.

[0030] The primer sequences for amplifying the LtSCRE11 fragment are:

[0031] pET32a-LtSCRE11-His-F:gctgatatcggatccgaattcATGACGCTCGACCCGTGC

[0032] pET32a-LtSCRE11-His-R:gtggtggtggtggtgctcgagTTTGCAAGAGTCGCTGCCG

[0033] 3. LtSCRE11 induces ROS burst and callose accumulation in Nicotiana benthamiana

[0034] We diluted the purified LtSCRE11 protein into a concentration gradient and injected it directly into Nicotiana benthamiana leaves. Figure 2 Figure A in the middle), LtSCRE11 purified protein at a concentration of 5μM and above can induce cell death. In order to determine whether LtSCRE11 can stimulate plant immunity, and to avoid the intensity of cell death induced by high concentrations of purified protein being too strong, thereby affecting the determination of other indicators, we used 5μM LtSCRE11 purified protein to treat Nicotiana benthamiana and peach seedling leaves to measure immune response-related indicators. First, Nicotiana benthamiana and peach seedling leaves were treated with flg22, LtSCRE11 and EV (blank vector), respectively, and the ROS burst was measured using the Luminol method. The results showed that LtSCRE11 can induce ROS burst in Nicotiana benthamiana and peach seedling leaves, both reaching a peak at around 30 minutes, while CK tended to calm down and no excitation peak was generated ( Figure 2 At the same time, the accumulation of ROS in tobacco leaves was detected by DAB staining. The results showed that after 6 hours of treatment with LtSCRE11 protein, the leaves of Nicotiana benthamiana showed obvious accumulation of brown-yellow ROS (as shown in Figure 2). Figure 2 (Figure C in the middle).

[0035] In addition, the aniline blue staining method was used to detect the induction of callose deposition in plants by LtSCRE11 protein. Similarly, 5μM LtSCRE11 protein was injected into Nicotiana benthamiana leaves for 6 hours, and the treated leaves were stained with aniline blue. Fluorescence microscopy revealed that a large amount of callose deposition could be observed in tobacco leaves treated with LtSCRE11 protein, while no callose deposition was observed in tobacco leaves treated with the control ( Figure 2 D and E). These results indicate that LtSCRE11 can induce ROS burst and callose accumulation.

[0036] 4. LtSCRE11 induces the expression of plant immunity-related genes

[0037] We injected 5 μM LtSCRE11 protein into leaves of Nicotiana benthamiana and peach seedlings, collected samples 6 h later, extracted total RNA using EASY spin Plus Plant RNA Rapid Extraction Kit (Aidlab, Beijing), tested RNA concentration and quality using Nanodropone (Thermo, USA), and used reverse transcription kit (RTK) to analyze the RNA. The RTReagent Kit with gDNAEraser (TaKaRa, Dalian, China) was used according to the instructions and reverse transcribed to obtain cDNA for RT-qPCR analysis. Figure 3 (Center Panel A) Immune-related genes in Nicotiana benthamiana, such as the pathogenesis-related protein genes NbPR1, 2, 3, and 4, the ethylene pathway-associated protein NbERF1, the jasmonic acid pathway-associated protein NbLOX, and the PTI marker genes NbCYP71D20, NbWRKY7, NbAcre31, and NbPTI5, were significantly upregulated. Furthermore, LtSCRE11 treatment also induced upregulation of the HR response marker genes NbHIN1 and NbHSR203J, indicating that the LtSCRE11-induced cell death response is a HR response.

[0038] In addition, up-regulated expression of pathogenesis-related protein genes PpPR1, 2, 3, 4 and PTI marker genes PpAcre31 and PpPTI5 was also detected in peach seedling leaves (e.g. Figure 3 These results indicate that LtSCRE11-induced cell death is a HR response and has PAMP elicitor activity, which can stimulate the transcriptional expression of genes related to plant immune pathways.

[0039] The primer sequences used for RT-qPCR are:

[0040] NbPR1-F CCGCCTTCCCTCAACTCAAC

[0041] NbPR1-R GCACAACCAAGACGTACTGAG

[0042] NbPR2-F AGGTGTTTGCTATGGAATGC

[0043] NbPR2-R TCTGTACCCACCATCTTGC

[0044] NbPR3-F GGCATTGGTTCTATTGT

[0045] NbPR3-R AATTTCTTTCCTACGGGCAGTATCATC

[0046] NbPR4-F GGCCAAGATTCCTGTGGTAGAT

[0047] NbPR4-R CACTGTTGTTTGAGTTCCTGTTCCT

[0048] NbERF1-F GGCGAATTTTCCGGGAGACT

[0049] NbERF1-R GGCTCCGATTTTACTTCGCC

[0050] NbLOX-F CCTTAAGGAGATGGAACT

[0051] NbLOX-R TCTAAGCTCATAAGCAATGG

[0052] NbHIN1-F CCAACTTGAACGGAGCCTATTA

[0053] NbHIN1-R AGGCATCCAAAGAGACACTAC

[0054] NbHSR203J-F ACGCAGATTTCAACCGAGTAT

[0055] NbHSR203J-R GCCAGTCGCATTGGAGATAA

[0056] NbCYP71D20-F AAGGTCCACCGCACCATGTCCTTAGAG

[0057] NbCYP71D20-R AAGAATTCCTTGCCCTTGAGTACTTGC

[0058] NbWRKY7-F CACAAGGGTACAAACAACACAG

[0059] NbWRKY7-R GGTTGCATTTGGTTCATGTAAG

[0060] NbAcre31-F AATTCGGCCATCGTGATCTTGGTC

[0061] NbAcre31-R GAGAAACTGGGATTGCCTGAAGGA

[0062] NbPTI5-F CCTCCAAGTTTGAGCTCGGATAGT

[0063] NbPTI5-R CCAAGAAATTCTCCATGCACTCTGTC

[0064] PpPR1-F TGACAAGGTGTGTGGGCATT

[0065] PpPR1-R CGGATCATAGTTGCACCCGA

[0066] PpPR2-F AAGTTTGAGGCAGCCAGTGA

[0067] PpPR2-R GGGTTCTCCAAGAGGTAGGC

[0068] PpPR3-F ACGGCTTAAACTCGCCAGAA

[0069] PpPR3-R TGATGCGGGCTTGAACTAGG

[0070] PpPR4-F GGTGACAAACACGGGCACAGGAG

[0071] PpPR4-R AAGAAGCGATCCCACTTTGAACT

[0072] PpAcre31-F TTGCCAAGCTAGGGTTCCAG

[0073] PpAcre31-R GGAGGGAACCAGAACAGTGG

[0074] PpPTI5-F CAAATGGCCTCGAACCCTCT

[0075] PpPTI5-R ATCGTATGCCAAGGCAGCTT

[0076] 5.LtSCRE11 is the MAPK system

[0077] MAPK activation experiments were performed according to Zhang Lei (2024). Leaves from 4-week-old Nicotiana benthamiana or 3-week-old peach seedlings were punched with a 1-cm diameter hole punch. Eighteen leaf discs were punched from 18 leaves. The leaf discs were placed in a 6-well plate, added with 4 mL of sterile water, and allowed to thaw overnight at room temperature in the dark. The sterile water was then replaced with 200 nM flg22 or 5 μM LtSCRE11 for treatment for 10, 20, and 30 min, respectively. The treated leaf samples were quickly blotted with absorbent paper to remove any residual liquid and stored frozen in liquid nitrogen at -80°C. Total protein was extracted using a heat lysis extraction buffer according to (Wang et al. 2023b), consisting of 20 mM Tris-HCl, pH 8.0, 5 mM ethylenediaminetetraacetic acid (EDTA), 1% SDS (w / v), 1% proteinase inhibitor cocktail (ABclonal, China), and 1 mM PMSF (Solarbio, China). The phosphorylation level of MAPKs proteins was detected by Western blot. The blocking solution was 1×TBST containing 5% BSA, the primary antibody was anti-p44 / 42 MAPK (Cell Signaling Technology, USA, 9101s, 1:2,000), and the secondary antibody was anti-Rabbit-HRP (ABclonal, China, AS014, 1:5,000).

[0078] The experimental results showed that LtSCRE11, like flg22, could activate the phosphorylation of MPK3 and MPK6 in leaves of Nicotiana benthamiana and peach seedlings at 10, 20 and 30 min after treatment ( Figure 4 Combined with the above experimental results, LtSCRE11 can not only induce ROS accumulation, callose deposition, and significant upregulation of immune-related genes, but also activate MAPK phosphorylation, further confirming that LtSCRE11 has the function of a PAMP and that the plant immune response induced by it is a typical PTI process.

[0079] 6. LtSCRE11 induces disease resistance in Nicotiana benthamiana and peach

[0080] To determine whether LtSCRE11 can induce plant resistance to pathogens, whole leaves of Nicotiana benthamiana and peach seedlings were injected with 5 μM LtSCRE11 protein, with empty protein as a control. Six hours after injection, leaves of Nicotiana benthamiana and peach were inoculated with the pathogen P. capsici LT263 and the pathogen L. theobromae JMB122, respectively. Forty-eight hours after inoculation, samples were collected and quickly frozen in liquid nitrogen at -80°C. Genomic DNA was extracted from 0.2 g of each sample. Genomic DNA from Nicotiana benthamiana was represented by the NbEF1α gene, and from P. capsici by the Pcβ-actin gene (Sheng et al. 2024). Genomic DNA from peach was represented by the PpEF1α gene, and from the pathogen L. theobromae by the LtITS gene. qPCR was used to detect the relative biomass of Phytophthora capsici infecting Nicotiana benthamiana leaves (pathogen P. capsici Pcβ-actin gene relative to Nicotiana benthamiana NbEF1α gene), and the relative biomass of L. theobromae infecting peach shoots (pathogen L. theobromae ITS gene relative to peach PpEF1α gene). The primer sequences used for qPCR are as follows:

[0081] Pcβ-actin-F:ACTGCACGTTCCAGACGATC

[0082] Pcβ-actin-R:CCACCACCTTGATCTTCATG

[0083] NbEF1α-F:TTGCTTGCTTTCACCCTTGG

[0084] NbEF1α-R:TCGAAACCAGAGATGGGGAC

[0085] LtITS-F:TCTCTTGGTTCTGGCATCGA

[0086] LtITS-R:TAATGACGTCTCGAACAGGCA

[0087] PpEF1α-F:TCCAGTTCTTGATTGCCACA

[0088] PpEF1α-R: CCATACCTGCATCTCCGTTC

[0089] In addition, the area of ​​lesions was counted to evaluate disease resistance. The results showed that compared with the control, the area of ​​lesions caused by pepper phytophthora (P. capsici) on Nicotiana benthamiana leaves after injection with LtSCRE11 was significantly smaller than that of the control, and the relative biomass was also significantly lower than that of the control ( Figure 5 Similarly, the area of ​​lesions caused by the gummogenic fungus (L. theobromae) on the leaves of peach seedlings was significantly smaller than that of the control treatment, and the relative biomass was also significantly smaller than that of the control treatment ( Figure 5 These results indicate that LtSCRE11 can enhance the disease resistance of Nicotiana benthamiana and peach to pathogens.

[0090] The foregoing is an example of the best mode of carrying out the present invention. Any portion not described in detail herein is common knowledge within the skill of one of ordinary skill in the art. The scope of protection of the present invention is determined by the claims. Any equivalent transformation based on the technical teachings of the present invention is also within the scope of protection of the present invention.

Claims

1. Application of LtSCRE11 protein in regulating resistance to peach gummosis, characterized in that: The LtSCRE11 protein is selected from any one of the following (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1; (b) A protein derived from (a) having the same enzymatic activity as that in (a) wherein the amino acid sequence is substituted, deleted or added with one or more amino acid sequences.

2. The use according to claim 1, characterized in that The specific application method is: use LtSCRE11 protein injection to treat peach tree leaves.

3. The use according to claim 2, characterized in that The LtSCRE11 protein is a purified LtSCRE11 protein diluted to a concentration of no more than 5 μM.

Images