Application of PpLecRK-IX.1 gene in improving resistance of peach trees to gummosis

Abstract

The invention relates to an application of a PpLecRK-IX.1 gene in improving the resistance of peach trees to gummosis, which is characterized in that a phytolectin receptor-like kinase gene PpLecRK-IX.1 is screened from peaches by using a yeast two-hybrid library by taking a pathogen-related molecular pattern (PAMP) protein secreted by peach gummosis pathogenic bacteria as a bait. Through agrobacterium tumefaciens-mediated transient overexpression, the gene is overexpressed in peach seedlings, and the resistance of leaves of the peach seedlings to peach gummosis is obviously enhanced; the target gene is silent in peach seedlings by using a VIGS technology, the resistance is obviously weakened, and the gene is proved to play an important role in peach gummosis resistance and can be used as a molecular marker or a gene resource to improve the resistance of peach trees to gummosis.

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 PpLecRK-IX.1 gene in improving the gummosis resistance of peach trees. Background Art

[0002] Peach gummosis is a major disease that seriously threatens the peach industry in my country's Yangtze River Basin and areas south of it. Its unclear pathogenic mechanism limits the development of prevention and control technologies. The causes of peach gummosis are complex, and there are currently no resistant varieties or effective prevention and control methods. In plant-fungus interactions, the PTI (PAMP-triggered immunity) response is the primary battlefield for plants to respond to the invasion of foreign microorganisms. Pattern recognition receptors located on the cell membrane surface recognize PAMPs ligands from microorganisms and activate a series of immune responses. Effector proteins secreted by certain pathogens can manipulate the immune function of host plants by stimulating host plant resistance or increasing sensitivity. Therefore, using pathogen effector proteins to screen target receptor proteins in plants is one of the important means of precise prevention and control of gummosis.

[0003] Plant lectin receptor-like kinases (LecRKs) are composed of an extracellular lectin domain, a transmembrane region, and an intracellular kinase domain. Plant LecRKs are a large subfamily of plant receptor kinases and play important roles in plant growth and development and in defense against pathogen invasion. LecRKs participate in plant immune signaling and play a key role. For example, Arabidopsis thaliana LecRK-I.9 regulates the jasmonic acid (JA) signaling pathway. Mutants of lecrk-I.9 impair cell wall defense and JA signaling, increasing susceptibility to the pathogens Plasmodiophora brassicae and Pseudomonas syringae pv. tomato (Pst) DC3000. Overexpression of PpLecRK-I.9 enhances Arabidopsis resistance to these pathogens (Bouwmeester et al. 2011, Balagué et al. 2017). At present, there are few studies on LecRKs in model plants, and there are no reports on LecRKs disease resistance in peach. Therefore, revealing the function of LecRKs in peach trees' resistance to gummosis infection is of great significance for the prevention and control of gummosis. Summary of the Invention

[0004] In order to solve the above problems, the present invention adopts the following technical solutions:

[0005] Application of PpLecRK-IX.1 gene in regulating resistance of peach to peach gummosis, wherein the PpLecRK-IX.1 gene is a nucleotide sequence capable of encoding a protein of (a) or (b) below:

[0006] (a) a protein consisting of an amino acid sequence shown in SEQ ID NO: 2;

[0007] (b) a protein derived from (a) by substitution, deletion or addition of one or more amino acid sequences in the amino acid sequence of (a) and having the same enzyme activity.

[0008] Further, the nucleotide sequence of the PpLecRK-IX.1 gene is selected from any one of (a) or (b) below:

[0009] (a) a DNA sequence with a nucleotide sequence shown in SEQ ID NO: 1;

[0010] (b) a nucleotide sequence having more than 90% homology with the DNA sequence of (a) and encoding the protein of claim 1.

[0011] Application of the primer of the PpLecRK-IX.1 gene in screening peach plants resistant to peach gummosis.

[0012] Application of the expression vector and the engineering bacteria of the PpLecRK-IX.1 gene in improving the resistance of peach to peach gummosis.

[0013] Further, the engineering bacteria include, but are not limited to, Escherichia coli and Agrobacterium.

[0014] A method for improving the resistance of peach to peach gummosis, wherein the PpLecRK-IX.1 gene is transiently overexpressed in the peach tree.

[0015] The present application has the following beneficial effects:

[0016] With the effector protein secreted by the gummosis bacteria as a bait, we screened a plant lectin receptor kinase gene PpLecRK-IX.1 through a yeast two-hybrid library, and found that the resistance of the leaves of the peach seedlings to peach gummosis was significantly enhanced through Agrobacterium-mediated transient overexpression of the gene, and the resistance was weakened through silencing of the target gene in the peach seedlings by VIGS, which proves that the gene plays an important role in the resistance of peach to gummosis and can be applied to improve the resistance of peach to gummosis.

[0017] The present application will be described in detail below with reference to the accompanying drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Figure 3 is a functional domain analysis diagram of PpLecRK-IX.1.

[0019] Figure 2 Figure 1 is a phylogenetic tree of PpLecRK-IX.1 (Prupe.4G175100.1) and its homologous proteins in different plant species;

[0020] Figure 3 Figure 4 is a schematic diagram of PpLecRK-IX.1 protein expression in ex vivo peach leaves overexpressing PpLecRK-IX.1, lesion area after inoculation with the pathogenic bacteria, expression of immune-related genes, and relative biomass of the pathogenic bacteria;

[0021] Figure 4 Figure 5 is a photograph of peach plants injected with PpPDS and empty vector in a VIGS silencing experiment;

[0022] Figure 5 Figure 6 is a schematic diagram of the relative expression of PpLecRK-IX.1 gene in ex vivo peach leaves in which PpLecRK-IX.1 is silenced, lesion area after inoculation with the pathogenic bacteria, expression of immune-related genes, and relative biomass of the pathogenic bacteria. DETAILED DESCRIPTION

[0023] The principles and features of the present application are described below in conjunction with the accompanying drawings, which are only used to explain the present application and are not intended to limit the scope of the present application.

[0024] Main experimental methods:

[0025] 1. Cloning and bioinformatics analysis of PpLecRK-IX.1

[0026] The full-length sequence of PpLecRK-IX.1 CDS (2112 bp) was cloned from peach cDNA, and the name in the peach genome is Prupe.4G175100.1. The functional domain of PpLecRK-IX.1 was analyzed using the online website InterPro (https: / / www.ebi.ac.uk / interpro / ), and visualized using IBS2.0. The results showed that PpLecRK-IX.1 encodes a protein of 703 amino acids, which contains an N-terminal signal peptide, an extracellular domain (ECD), a transmembrane region, and a cellular domain (CD) Figure 1). To construct the phylogenetic tree of PpLecRK-IX.1 homologous proteins, we searched the conserved domain of L-LecRKs in different representative plant group genomes using RPS-BLAST (E-value of 0.01) and performed multiple sequence alignment using MAFFT. The phylogenetic tree was constructed by the maximum likelihood method of GeneRax (Morel et al 2020) and IQ-TREE (auto-exhibit value set to 1000), and visualized using ggtree Figure 2

[0027] The full-length sequence of the PpLecRK-IX.1 gene CDS is:

[0028] ​

[0029] The PpLecRK-IX.1 amino acid sequence is as follows:

[0030] MVVLLLLLL FHLAPCATSL NFNFSTFPNG INNLSFEGDA FVDGAFLRL

[0031] TKSAADDVQD QSVGRATYSQ PFLLRDNATG KLADFTTNFT FTINSLDKTP

[0032] YADGLAFFLA PNGSALNRTI GRGGSLGLPT INPEKNESTN LYPFVAVEFD IF

[0033] QNAVTSVQDP AGDHVGIDVN SKVTRAWNGS ITQGRDNNAW IRYDSG

[0034] SKNLSVTFTTY ENGVWVRRYF DYMVDVNEIL QGWVIVGFSA ATGAMTA

[0035] LHKINSWSFN STSLIDENAK NNTPVAPEPT PIVEPESGNG INIGLVVGLV VG

[0036] GCVLLVGGFC LVWFIFWKKG GTGESSDNDE DPMVNDPIDD EFEKGTGPK

[0037] KFSYKILAQSTNDFDEGEKLGEGGFGGVYRGFVKDLNSYVAVKRVSSGSR

[0038] QGLKEYAAEVRIISRLRHRNLVQLIGWCHEKRELLLVYEFMSNGSLDSHL

[0039] FKGKSLLGWE ARYRIAQGLA SGLFYLHEEW EQCVLHRDIK SSNIMLDSNF

[0040] NAKLGDFGLA RLVDHGKQSQ TVLAGTMGYM APECVTTGKA SKETDVY

[0041] SFGVVALEIA CGRKPIDPKL GSTSKINMVE WVWELYGQG KVIEAADPKL CG

[0042] DFDEKQIECLLIVGLWCAHPDYKIRPSTQQIIQVLSLEVAPPILPSKMPIASY

[0043] FSPPVSFSILSGDVTGSERGQTESLSYGYNTNSSQFTSSSASNSSPSASLLYT

[0044] N (SEQ ID NO: 2)

[0045] 2. Construction of overexpression vector

[0046] Using one-step cloning technology, the pSAK277-HA vector plasmid was first linearized by enzyme cutting (enzyme cutting site was Kpnl); according to the design of the homologous arm primer of the full-length coding sequence of PpLecRK-IX.1, the target fragment was amplified, and the product was recovered by cutting gel and connected to the pSAK277-HA vector by ClonExpress IIOne Step Cloning Kit (Vazyme, China) kit; the ligation product was transformed into DH5a competent cells, and the vector resistance was spectinomycin (Spec) resistance, PCR detection of colonies, according to the agarose gel electrophoresis, the colonies with correct size bands were shaken and the plasmid was extracted, and the extracted plasmid was sent to the company for sequencing. After sequencing, the correct alignment analysis was carried out by SnapGene software, and the correct alignment of the bacterial liquid was extracted and stored at -20℃ for standby.

[0047] The primer sequence of the amplified fragment is:

[0048] pSAK277-PpLecRK-IX.1-HA-F: actagtggatccaaagaattcATGGTTGTCCTCCTTCTTCTTCTC pSAK277-PpLecRK-IX.1-HA-R: gtatgggtactcgagaagcttATTTGTGTACAAAAGTGAAGCTGATG

[0049] The primer sequence of the positive identification is:

[0050] pSAK277-HA-F: GAAGACCAAAGGGCTATTGAGAC

[0051] pSAK277-HA-R: GATGTCGCTATAAACCTATTCAGCA

[0052] 3. Agrobacterium-mediated transient overexpression of peach seedlings

[0053] The recombinant plasmids pSAK277-GUS and pSAK277-PpLecRK-IX.1 obtained in step 2 were transformed into GV3101 competent cells, and positive colonies were picked and shaken overnight in 1 mL LB (containing guanyin and rifampicin) liquid culture medium at 220 rpm and 28°C; the shaken bacterial solution was pipetted into 50 mL LB (containing guanyin and rifampicin) at a ratio of 1:100 and cultured for 16 hours; the bacterial solution was collected into a centrifuge tube and the precipitate was collected by centrifugation; the precipitate was washed twice with MMA buffer and the OD600 of the bacterial solution was adjusted to 1.2, and it was allowed to stand at room temperature in the dark for 2 hours; the bacterial solution was injected from the back of the leaf into the fully expanded leaves of peach seedlings of about 4 weeks old using a 1 mL disposable medical syringe with the needle removed until the entire leaf appeared water-soaked. After the injection, it was placed in a growth room for 2 days.

[0054] 4. Identification of resistance of peach seedlings transiently overexpressing PpLecRK-IX.1 to L. theobromae JMB122

[0055] Using the injected peach detached leaves as materials, the leaves were inoculated with the peach gummosis pathogen L. theobromae JMB122 according to the method of Zhang Dongmei (2021). The symptoms were observed and photographed 1 and 2 days after inoculation; the lesion area was then measured using ImageJ software. The leaves 2 days after inoculation were blotted dry with absorbent paper to remove the residual liquid, and then frozen in liquid nitrogen and stored in a -80°C refrigerator. The leaves were ground into powder with liquid nitrogen and total RNA was extracted using the EASY spin Plus Plant RNA Rapid Extraction Kit (Aidlab, Beijing). The RNA concentration and quality were tested using Nanodrop one (Thermo, USA). The reverse transcription kit was used to extract the total RNA. The Reagent Kit with gDNAEraser (TaKaRa, Dalian, China) was operated according to the instructions, and cDNA was obtained by reverse transcription. The cDNA concentration and quality were detected by Nanodrop one, and then qRT-PCR was performed. Quantitative primers were designed using the online website NCBI Primer-BLAST, and the peach PpTEF2 gene (Zhang et al 2023) and the peach gummosis pathogen L. theobromae LtTUBULIN gene (Zhang et al 2024a) were used as internal reference genes for data balancing. The RT-qPCR reaction system was 10 μL, referring to the operating manual of the kit. The reaction procedure was as follows: 95℃ for 5 min; 95℃ for 10 s, 60℃ for 20 s, and 72℃ for 20 s, repeated for 40 cycles. Three technical replicates were set for each sample, and the experiment was repeated 3 times. The relative expression data of genes were analyzed using 2 -ΔΔCTLivak and Schmittgen 2001). The PpEF1a gene represents peach genomic DNA and the pathogen L. theobromae LtITS gene represents its genomic DNA. The relative biomass of L. theobromae in the invaded peach shoot tissues (pathogen L. theobromae LtITS gene relative to peach PpEF1a gene) was detected by qPCR.

[0056] The quantitative primer sequences used are as follows:

[0057] qPCR-PpLecRK-IX.1-F: CCC CCCATTGTTGAACCCGAG

[0058] qPCR-PpLecRK-IX.1-R: GCTACTTTCCCCTGTTCCCC

[0059] PpTEF2-F: AGCAAGTCACCCAACAAGCATA

[0060] PpTEF2-R: CCAACCAAACTCTTCAGCCAAT

[0061] LtTUB-F: AATCGGTGCTGCTTTCTGG

[0062] LtTUB-R: TTGTTGGACGCCTCGTTG

[0063] LtITS-F: TCTCTTGGTTCTGGCATCGA

[0064] LtITS-R: TAATGACGCTCGAACAGGCA

[0065] PpEF1a-F: TCCAGTTCTTGATTGCCACA

[0066] PpEF1a-R: CCATACCTGCATCTCCGTTC

[0067] The results showed that the lesion area of the leaves overexpressing PpLecRK-IX.1-HA was reduced by 79.54% and 55.57% at 24 and 48 h after inoculation, respectively, which were significantly smaller than the control group ( Figure 3 , A, B, D, E); Western blot results showed normal expression of PpLecRK-IX.1 protein and control GUS protein ( Figure 3 , C); the relative biomass of pathogen in the leaves transiently overexpressing PpLecRK-IX.1-HA was also significantly lower than the control group at 48 h after inoculation ( Figure 3, F). Meanwhile, the relative transcription levels of PpPR2 and PpPR4 in PpLecRK-IX.1-HA leaves with transient overexpression were significantly higher than those in the control group at 48 h after inoculation ( Figure 3 , G, H). The above results showed that transient overexpression of PpLecRK-IX.1 could significantly enhance the resistance of peach seedlings to P. prunicola.

[0068] 5. VIGS-mediated PpLecRK-IX.1 gene silencing in peach seedlings

[0069] Virus-mediated silencing of peach seedlings was referred to (Cui et al 2020, Zhang et al 2023) and the specific method was as follows: PpLecRK-IX.1 gene silencing fragment (200 bp) was designed using VIGS online tool (https: / / vigs.solgenomics.net / ), and specific primers were designed according to the fragment to construct into vector pCaRNA3 (Xba I enzyme site) and transformed into Agrobacterium strain GV3101. The pCaRNA3 with the target fragment and the helper vector pCaRNA1 & 2 were adjusted to OD 600 1, mixed at 1:1 equal volume, and incubated at room temperature in the dark for 3 h. Healthy peach seedlings growing to 3 weeks old were selected for injection. The mixed bacterial solution of pCaRNA3-PpPDS and pCaRNA1 & 2 was used as a positive control, and pCaRNA3 and pCaRNA1 & 2 were used as blank controls. After the positive control new leaves showed obvious white phenomenon along the main leaf vein (Fig. Figure 4 , B), indicating that the silencing time was sufficient for subsequent experiments. Photographs were taken and the silencing efficiency was detected by RT-qPCR.

[0070] The sequence of PpLecRK-IX.1 gene silencing fragment is as follows:

[0071] TCCCGCCGGCGATCATGTTGGTATTGATGTCAACTCTGTCAAGTCT

[0072] AAGGTTACAAGGGCGTGGAATGGTAGTATTACACAGGGACGAGACAAT

[0073] AATGCTTGGATTCGTTACGATTCTGGATCCAAAAATCTTAGCGTTACATT

[0074] TACTACTTATGAAAATGGTGTCTGGGTTAGAAGGTATTTTGATTACATGG

[0075] TTGATG

[0076] The sequence of the fragment amplification primer is as follows:

[0077] pCaRNA3-PpLecRK-IX.1-F:

[0078] aagacctgcttgagatctagaGGTTACAAGGGCGTGGAATG

[0079] pCaRNA3-PpLecRK-IX.1-R:

[0080] gtgtgcttatctcactctagaTCAACCATGTAATCAAAATACCTTCTAA

[0081] The sequence of the primer used for RT-qPCR is as follows:

[0082] qPCR-PpLecRK-IX.1-F: CCCCCATTGTTGAACCCGAG

[0083] qPCR-PpLecRK-IX.1-R: GCTACTTTCCCCTGTTCCCC

[0084] RT-qPCR detection showed that the transcription level of PpLecRK-IX.1 in PNRSV-PpLecRK-IX.1 silenced plants was significantly reduced by 49.21% compared with the PNRSV-EV control group, indicating that PpLecRK-IX.1 was effectively silenced Figure 5 , C).

[0085] 6. Resistance identification of silenced peach plants to L. theobromae JMB122

[0086] After inoculation of L. theobromae JMB122, it was found that the lesion area of PNRSV-PpLecRK-IX.1 silenced plants was significantly larger than that of the control group 24 and 48 h after inoculation, with an increase of 176.13% and 67.18%, respectively Figure 5 , A, B, D, E); 48 h after inoculation, the above inoculated leaves were ground into powder with liquid nitrogen, and the methods for extracting RNA and DNA, determining the relative expression of genes and the relative biomass were the same as those in 4. The results showed that the relative biomass of the pathogen in the leaves of PNRSV-PpLecRK-IX.1 silenced plants was also significantly higher than that of the control group Figure 5 , F); the relative transcription levels of PpPR2 and PpPR4 in the leaves of PNRSV-PpLecRK-IX.1 silenced plants were significantly lower than those of the control group 48 h after inoculation Figure 5, G, H). These results indicate that transient silencing of PpLecRK-IX.1 significantly increased the susceptibility of peach to Gummosis spp. Combining the results of transient overexpression and silencing experiments, PpLecRK-IX.1 positively regulates peach resistance to Gummosis spp.

[0087] The primer sequences used for fluorescence quantification are:

[0088] PpPR2-F:AAGTTTGAGGCAGCCAGTGA

[0089] PpPR2-R:GGGTTCTCCAAGAGGTAGGC

[0090] PpPR4-F: GGTGACAAACACGGGCACAGGAG

[0091] PpPR4-R:AAGAAGCGATCCCACTTTGAACT

[0092] 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 the PpLecRK-IX.1 gene in regulating resistance to peach gummosis, characterized in that: The PpLecRK-IX.1 gene is a nucleotide sequence capable of encoding the following (a) or (b) protein: (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2; (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 nucleotide sequence of the PpLecRK-IX.1 gene is selected from any one of the following (a) or (b): (a) the nucleotide sequence is a DNA sequence as shown in SEQ ID NO: 1; (b) A nucleotide sequence that has 90% or greater homology with the DNA sequence in (a) and encodes the protein of claim 1.

3. Use of primers for detecting the PpLecRK-IX.1 gene of claim 1 in screening plants resistant to peach gummosis.

4. Use of an expression vector and an engineered bacterium containing the PpLecRK-IX.1 gene according to claim 1 in improving the resistance of peach to gummosis.

5. The use according to claim 4, characterized in that The engineered bacteria include but are not limited to Escherichia coli and Agrobacterium.

6. A method for improving peach resistance to peach gummosis, characterized in that: Transient overexpression of the PpLecRK-IX.1 gene in peach trees.

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