BcSPS1 gene and its application in reducing pathogenicity of botrytis cinerea

Abstract

The BcSPS1 gene and application thereof in reducing pathogenicity of Botrytis cinerea belong to the field of microbial genetic engineering technology, and the BcSPS1 gene provided by the application has a DNA sequence as shown in SEQ ID No:1 and is composed of 1947 nucleotides; the protein encoded by the BcSPS1 gene has an amino acid sequence as shown in SEQ ID No:2 and is composed of 612 amino acids; the BcSPS1 gene can be applied in the field of plant Botrytis cinerea genetic engineering; and the BcSPS1 gene for controlling pathogenicity of Botrytis cinerea can be knocked out to make the pathogenicity of Botrytis cinerea defective and can be applied in reducing pathogenicity of Botrytis cinerea.

Description

Technical Field

[0001] This invention belongs to the field of microbial genetic engineering technology, specifically relating to the discovery of new genes controlling fungal pathogenicity in the field of plant protection and the application of their encoded proteins. Background Technology

[0002] Botrytis cinerea, commonly known as gray mold, is a fungus belonging to the phylum Ascomycota. It is the pathogen of gray mold and can infect more than 1400 plant species, including almost all vegetables and fruit trees. The disease can occur from the seedling stage, fruiting stage, and storage stage. Furthermore, all parts of the plant can be infected by Botrytis cinerea. Typical symptoms on leaves include "V"-shaped lesions; flowers mainly show rot and wilting; and fruits mainly show rot and fruit drop. The occurrence and spread of the disease are closely related to environmental humidity and temperature, with severe outbreaks occurring at 20℃-23℃ and relative humidity above 90%. Therefore, gray mold is a disease that thrives in low-temperature and high-humidity conditions, making it highly susceptible during rainy seasons or in protected cultivation. The economic losses caused by this disease worldwide each year range from $100 to $1000 billion. Due to its wide host range, serious damage to production, and the maturity of related molecular research techniques, Botrytis cinerea has become one of the most important model plant pathogenic fungi and has been extensively studied.

[0003] Gray mold is a typical necrotrophic pathogenic fungus that produces various pathogenic factors involved in its pathogenesis. These factors mainly include cell wall degrading enzymes, cutinases, toxins, plant hormones, enzymes that resist host responses, small RNAs, and small molecules. These factors work together to enable gray mold to kill host cells and decompose dead host tissue for nutrients. Under natural conditions, gray mold primarily uses conidia as the primary and secondary source of infection. Gray mold often overwinters and oversummers in diseased plant debris as mycelium, conidia, or sclerotia, becoming the primary source of infection for the next growing season. When conditions are suitable, sclerotia germinate to produce mycelium and conidiophores, and generate a large number of conidia. Mature conidia can be spread by wind, rainwater, irrigation water, and agricultural operations. Under low temperature and high humidity conditions, conidia germinate to form germ tubes. The ends of the germ tubes swell slightly and develop into appressoriums or further form infection pads and other infection structures, which mainly invade from decaying flower parts, wounds and necrotic tissues.

[0004] When high concentrations of *Botrytis cinerea* conidia infect the host, disease development is rapid, primarily through appressoriums formed at the germ tube tip. As spore concentration decreases, the proportion of invasion via germ tube tip decreases, and the disease progression is delayed by 1-4 days. At this stage, invasion mainly occurs through appressoriums or infection pads developed from hyphae. After invading host cells, *Botrytis cinerea* directly faces the challenge of a hostile environment within the host tissue. The pathogen must rapidly adjust, suppressing the plant's defense response while actively adapting to the physical and chemical environment within the host cells. Only by achieving both can *Botrytis cinerea* successfully parasitize plants. To a large extent, *Botrytis cinerea* achieves this by altering its metabolic pathways and secreting related effector factors (such as toxins). However, the genes, proteins, and metabolites involved in these processes, as well as the molecular mechanisms of their regulation, remain poorly understood. In-depth research in this field, identifying the key factors that gray mold uses to adapt to the host's internal environment, will not only help reveal the molecular mechanism of this necrotrophic pathogenic fungus, but may also lead to the discovery of proteins that can serve as targets for fungicides, laying the theoretical and technical foundation for the development of highly effective agents to control gray mold and other similar diseases.

[0005] The SPS1 gene encodes spermine synthase, which catalyzes the synthesis of spermine. In the semi-vitreous plant pathogen *Blastomyces oryzae*, SPS1 participates in the pathogen's invasion of the host plant. The SPS1 gene also exists in the necrotrophic pathogen *Botrytis cinerea*, but its function has not yet been identified. Analyzing the pathogenic function of the *Botrytis cinerea* SPS1 gene and evaluating its role in the development and pathogenesis of *Botrytis cinerea* will help identify potential control targets and screen novel fungicides against *Botrytis cinerea*. Summary of the Invention

[0006] The purpose of this invention is to provide a gene and its encoded protein that controls the pathogenicity of gray mold.

[0007] The pathogenicity control gene provided by this invention is derived from *Botrytis cinerea*, named BcSPS1, and its DNA sequence is shown in SEQ ID No:1. This DNA sequence is the open reading frame of the BcSPS1 gene, consisting of 1947 nucleotides, including three exons located between nucleotides 1 to 1076, 1133 to 1433, and 1486 to 1947 from the 5' end of SEQ ID No:1, respectively. The total coding region length is 1839 nucleotides.

[0008] The present invention provides a protein encoded by the BcSPS1 gene, the amino acid sequence of which is shown in SEQ ID No:2, and the sequence consists of 612 amino acids.

[0009] The BcSPS1 gene, derived from the pathogenicity control fungus *Botrytis cinerea*, can be applied to the field of genetic engineering for plant resistance to *Botrytis cinerea*.

[0010] The BcSPS1 gene, which controls pathogenicity from Botrytis cinerea, can be knocked out to render it defective, and this can be used to reduce the pathogenicity of Botrytis cinerea.

[0011] This invention demonstrates that the deletion of the BcSPS1 gene significantly reduces the pathogenicity of *Botrytis cinerea*, indicating that the BcSPS1 gene is essential for *Botrytis cinerea* to cause gray mold disease in crops. Therefore, screening for compounds that can inhibit the expression of this gene and the expression, modification, and localization of its encoded protein can effectively control the occurrence of gray mold, thereby contributing to the development of novel fungicides. One important application of the BcSPS1 gene provided in this invention is that the expression of this gene and the expression, modification, and localization of its encoded protein product can serve as important candidate target sites for reducing the pathogenicity of *Botrytis cinerea*. Attached Figure Description

[0012] Figure 1 A schematic diagram illustrating the domain analysis of the protein encoded by the BcSPS1 gene.

[0013] Wherein: the left arrow indicates the transmembrane region; the right arrow indicates the spermine / spermine synthase domain.

[0014] Figure 2 This is a schematic diagram of the knockout strategy of the BcSPS1 gene of Botrytis cinerea (gene replacement through homologous recombination);

[0015] Among them: B05.10 is the wild-type strain of Botrytis cinerea, pSPS1-KO is the knockout vector, and ΔBcsps1 is the knockout mutant of the BcSPS1 gene; primers P1, P2 and P5, P6 are used to amplify the upstream and downstream sequences of the BcSPS1 gene, respectively, and serve as homologous arms of the knockout vector; primers P3, P4, P7 and P8 are used to verify the mutant and complementary strain.

[0016] Figure 3 Electrophoresis diagrams for PCR verification of BcSPS1 gene knockout mutants and genetically complementary strains;

[0017] Among them, P7, P8, P3, and P4 are the primers used, and their corresponding binding sites are shown in [reference needed]. Figure 2 M is a BcSPS1 gene knockout mutant; ΔBcsps1-C is a complementary strain of mutant M with the complete BcSPS1 gene introduced; the DNA molecular standard used is DL2000.

[0018] Figure 4Photographs comparing the pathogenicity of BcSPS1 gene knockout mutants and wild-type strains;

[0019] The selected host was the "Spring Peach" tomato, and the method of inoculating the fruit with fungal cakes was adopted. Evaluation was carried out 5 days after inoculation; WT was the wild-type strain B05.10 of gray mold, and M was a knockout mutant of the BcSPS1 gene.

[0020] Figure 5 A schematic diagram illustrating the quantitative analysis of lesion size produced by mutant and control strains of the BcSPS1 gene infecting the host.

[0021] The inoculation method was the same as above. Measurements were taken 5 days after inoculation and converted into relative values ​​to compare the pathogenicity of each strain. *** indicates a significant difference at the p<0.001 level.

[0022] Figure 6 Photographs comparing the pathogenicity of the BcSPS1 gene knockout mutant with that of the wild-type strain and the complementary strain ΔBcsps1-C.

[0023] The selected host was common bean, and the method of inoculating detached leaves with mycelial cakes was used. Evaluation was conducted 24 h and 48 h after inoculation; all strains used are indicated in the figure.

[0024] Figure 7 A schematic diagram illustrating the quantitative analysis of lesion size produced by mutant, control, and complementary strains of the BcSPS1 gene infecting the host.

[0025] The inoculation method was the same as above. Measurements were taken 24 hours and 48 hours after inoculation and converted into relative values ​​to compare the pathogenicity of each strain. *** indicates a significant difference at the p<0.001 level. Detailed Implementation

[0026] To better describe the present invention, specific embodiments are provided below. Unless otherwise specified, the methods in the following embodiments are conventional methods.

[0027] Example 1: Correlation analysis of the BcSPS1 gene

[0028] The open reading frame (ORF) of the *Botrytis cinerea* BcSPS1 gene consists of 1947 nucleotides and contains 3 exons. The full-length cDNA coding region is 1839 nucleotides, encoding a protein product of 612 amino acids. Domain analysis revealed that the C-terminus of the BcSps1 protein contains a spermine / spermine synthase domain, and its N-terminus contains five transmembrane regions (see...). Figure 1 Therefore, BcSps1 is a membrane-bound protein.

[0029] Example 2: Knockout of the BcSPS1 gene

[0030] 1) Construction of the knockout vector

[0031] Primers P1 (5'-AACTCGAGGGTGGGCTCGCATGTCGAGATGAAGGCAGC-3') and P2 (5'-CTCGAATTCCCACTAGTGAATGCGACAATTGTTTTGTTTGAG-3') were used to amplify the upstream 630 bp fragment of the BcSPS1 gene using genomic DNA from *Botrytis cinerea* strain B05.10 as a template. Primers P5 (5'-AGTCGACCTGCAGGCATGCATAATCACATAACCTCCTTCCTATTATG-3') and P6 (5'-AATGCGGCTCCACAGCTGCAAGCCAGCCAATCCAAATTG-3') were used to amplify the downstream 630 bp fragment of the *Botrytis cinerea* BcSPS1 gene. The reaction system was 10 mmol / L dNTP. Mixture, 0.4 μL; 5×PCR buffer, 4 μL; forward and reverse primers, 1 μL each (10 μmol / mL); template DNA, 1 μL; Q5 high-fidelity enzyme, 0.2 μL; ddH2O, 12.4 μL; the amplification program was as follows: pre-denaturation at 98℃ for 30 seconds, then (1) denaturation at 98℃ for 10 seconds; (2) annealing at 68℃ for 30 seconds; (3) extension at 72℃ for 60 seconds; (4) 25 cycles; (5) extension at 72℃ for 2 minutes. The two DNA amplification products were cloned into the pXEH vector to construct the knockout vector pSPS1-KO (see Figure 2 ), and then performed sequencing verification.

[0032] The Botrytis cinerea strain B05.10 used in this invention was purchased from the Fungal Genetics Stock Center (FGSC). Others who require this strain can purchase it from the FGSC. Relevant preservation information is as follows:

[0033] Strain number: FGSC 10317.

[0034] Address of the depository: Fungal Genetics Stock Center, Department of Plant Pathology, Kansas State University, 4024 Throkmorton Plant Sciences Center, Manhattan, KS 66506 USA.

[0035] Website: http: / / www.fgsc.net / scripts / StrainSearchReturnPage.asp?OrgID= 23812 .

[0036] 2) Transformation of gray mold

[0037] a. Culture of Agrobacterium

[0038] Single colonies of *Agrobacterium tumefaciens* strain Agl-1 containing the binary vector pSPS1-KO were picked and inoculated into MM liquid medium (0.205% dipotassium hydrogen phosphate, 0.145% potassium dihydrogen phosphate, 0.015% sodium chloride, 0.05% magnesium sulfate heptahydrate, 0.01% calcium chloride hexahydrate, 0.00025% ferrous sulfate heptahydrate, 0.05% ammonium sulfate, 0.2% glucose) containing 50 μg / ml kanamycin and 10 μg / ml rifampin. The medium was incubated at 250 rpm and 28°C with shaking for 48 h. After centrifugation at 4000 rpm for 5 min, the supernatant was discarded, and the culture was transferred to IM liquid medium (0.205% dipotassium hydrogen phosphate, 0.145% potassium dihydrogen phosphate, 0.015% sodium chloride, 0.05% magnesium sulfate heptahydrate, 0.01% calcium chloride hexahydrate, 0.00025% ferrous sulfate heptahydrate, 0.05% ammonium sulfate, 0.2% glucose, 200 μM AS, MES). Resuspend in 0.854% glycerol (0.5%), centrifuge at 4000 rpm for 5 minutes, discard the supernatant; resuspend in IM medium, incubate at 28℃ and 250 rpm for 6 hours for pre-induction.

[0039] b. Sporulation culture of Botrytis cinerea

[0040] Strain B05.10 was selected. A small amount of spores were spread on PDA medium (20% boiled and filtered potato, 2% glucose, 1.5% agar) and incubated at 28℃ for 8 hours to allow rapid spore germination. The culture was then transferred to 20℃ for 3-5 days. After the cell surface was covered with gray spores, the spores were scraped and collected using 1M liquid medium and observed under a microscope. The spore concentration was adjusted to 3.5 × 10⁻⁶ using a hemocytometer. 5 per mL.

[0041] c. Co-culture of Agrobacterium tumefaciens and Botrytis cinerea conidia and screening of transformants

[0042] Equal volumes of Agrobacterium tumefaciens bacterial suspension and Botrytis cinerea spore suspension, pre-induced for 6 hours in IM liquid medium, were mixed. AS was added to achieve a final concentration of 500 μM. The mixture was thoroughly mixed, and then spread evenly at 250–350 μL / plate onto IM medium lined with cellophane. The mixture was incubated in the dark at 22°C for 48 hours. After co-culturing, the cellophane was transferred to PDA medium containing 100 μg / mL hygromycin, and incubated under the same conditions. After 4–7 days, expanded colonies were picked and transferred to selection medium containing the same antibiotic.

[0043] 3) Validation of knockout mutants

[0044] Transformants were screened by PCR amplification using two pairs of primers. Transformants with the following amplification results were identified as BcSP S1 gene deletion mutants: primer P7 (5'-TTGATGCCTGCCGAATGG-3') on the genome outside the upstream homologous arm amplified a recombinant fragment of the expected size (930 bp) when paired with primer P8 (5'-ACAGACGTCGCGGTGAGTTCA-3') for the hygromycin resistance gene; while primers P3 (5'-GATTCCGTGCTATGCGTTGT-3') and P4 (5'-CTCCCAGTCTCATTCCTCCTTA-3') for the coding region showed no amplification band (wild-type strains amplified a 958 bp fragment). As a result, the BcSPS1 gene deletion mutant strain M was screened from the transformants for subsequent functional analysis (see [link to relevant documentation]). Figure 3 ).

[0045] Example 3: Genetic Complementation of BcSPS1 Gene Knockout Mutant

[0046] Using primers CF (5'-TGGGAATTCGAGCTCGGTACCGATGTCAGTTCATTGAAATAC-3') and CR (5'-CCTTCAATATCAGTTGGTACGAAGGAGGTTATGTGATTAAG-3'), the full-length 2542 bp gene of *Botrytis cinerea* (containing the promoter, open reading frame, and terminator) was amplified and cloned into the pXEGb vector (containing the genistein resistance gene) to construct the genetic complementation vector pSPS1-ko-c. Sequencing verification confirmed the absence of amino acid mutations. Using the *Agrobacterium*-mediated transformation method described above, and screening with 50 mg / L genistein, the complementary fragment was transformed into the genome of the *BcSPS1* gene deletion mutant strain M, obtaining the genetically complementary strain ΔBcsps1-C. PCR amplification was performed using primers P3 and P4, which were previously used for mutant verification, and the results were as expected (see [link to relevant documentation]). Figure 3 Similar to mutant strain M, the original BcSPS1 gene in the complementary strain ΔBcsps1-C was replaced with the hygromycin resistance gene HPH, but it also had an additional BcSPS1 gene that was subsequently introduced (the amplification results of primers P3 and P4 in the coding region were also positive).

[0047] Example 4: The role of the BcSPS1 gene in the pathogenicity of Botrytis cinerea.

[0048] The pathogenicity of the BcSPS1 gene mutant was evaluated using the fruit inoculation method. "Chuntao" tomatoes of uniform size and maturity purchased from the market were selected and placed horizontally in a container. A 5.2 mm diameter mycelium disc of the test strain was picked and inoculated onto the fruit surface (the mycelium disc was taken from the vigorous peripheral mycelium after 3 days of cultivation on PDA medium). The mixture was incubated at 20℃ in the dark with humidity. The pathogenicity of the test strain was evaluated after 5 days. The results showed that the wild-type strain could cause disease on tomato fruits normally, damaging the fruit and creating wounds 3 days after inoculation. By 5 days, lesions covered half of the fruit surface. Although the knockout mutant strain could utilize the nutrients from the mycelium disc to produce mycelium, it could not invade the tomato fruit to create wounds, and it failed to induce disease in tomatoes even 6 days after inoculation (see...). Figure 4 , Figure 5 ).

[0049] This study further employed the more susceptible detached leaf inoculation method to evaluate the changes in pathogenicity of the BcSPS1 gene mutant. Mature leaves were collected from green bean plants grown in a greenhouse, placed horizontally in a container, and 5.2 mm diameter mycelium pellets of the test strain were picked and inoculated onto the leaf surface (the method for obtaining the mycelium pellets was the same as described above). The mixture was then incubated in the dark at 20°C under moist conditions, and the pathogenicity of the test strains was evaluated at 24 h and 48 h. The results showed that the BcSPS1 gene knockout mutant exhibited significantly delayed disease development compared to the wild type. Although lesions still formed, the lesion area was significantly smaller than that of the wild type, making it difficult for the lesions to expand. The pathogenicity of the complementary strain essentially recovered to the level of the wild type (see...). Figure 6 ). Measurements of leaf lesion area revealed that at 24 hours, wild-type and complementary strains had already developed lesions, but the BcSPS1 gene knockout mutant did not develop the disease. At 48 hours, the BcSPS1 gene knockout mutant developed the disease, but the lesion area caused by its infection was only about 15% of that of the wild type (see...). Figure 7 ).

[0050] The results of this invention indicate that the BcSPS1 gene is involved in the pathogenic process of gray mold on plants and is essential for gray mold to infect the host. If this gene or its encoded protein loses its activity, the ability of gray mold to infect the host and cause disease will be severely impaired or even completely lost.

Claims

1. A kind BcSPS1 Genes in reducing gray mold ( Botrytis cinerea Its application in pathogenicity, specifically through knockout BcSPS1 Genes to reduce pathogenicity, the BcSPS1 The DNA sequence of the gene is shown in SEQ ID No:1.

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