Glycoside hydrolase fsgh28c and its coding gene in regulating pathogenicity of bacterial strain

Abstract

The present application relates to the field of biotechnology, in particular to the application of glycoside hydrolase FsGH28c and its coding gene in regulating pathogenicity of a bacterial strain. The present application provides a glycoside hydrolase coding gene FsGH28c related to the development of Fusarium solani pisi, the utilization of carbon source and pathogenicity. After knocking out the FsGH28c gene of Fusarium solani pisi, the spore production of Fusarium solani pisi is reduced, the spore development is deformed, and the utilization rate of carbon source (especially pectin) is reduced. In addition, the amount of fungal colonization at the root of the host pepper and the pathogenicity of the FsGH28c gene deletion mutant are reduced. The FsGH28c gene of the present application positively regulates the pathogenicity of Fusarium solani pisi, and can be used as an important candidate gene for the study of the pathogenic mechanism of Fusarium solani pisi.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to the application of glycoside hydrolase FsGH28c and its coding gene in regulating pathogenicity of a bacterial strain. BACKGROUND

[0002] Fusarium wilt of pepper is a vascular disease caused by a soil-borne fungus Fusarium solani, which causes significant economic losses to the pepper industry. As a soil-borne pathogenic fungus, F. solani infects more than 100 host plants, and when it harms pepper, the disease often occurs in the adult stage, with yellowing and shedding of leaves from bottom to top, and vascular necrosis of the underground roots and the stem near the ground. Currently, there is a lack of effective fungicides on the market to effectively control the pathogen.

[0003] F. solani is a member of the F. solani species complex (FSSC) system, and belongs to a semi-vital pathogenic fungus. Its chlamydospores and mycelium can survive in the soil for a long time, making it difficult to control and seriously threatening the sustainable development of the pepper industry in China. When the environment of the pepper plantation is suitable, the chlamydospores or mycelium of F. solani remaining in the soil are induced to re-germinate by root exudates, and then invade the pepper roots through the root tips or root wounds. In order to complete the infection, the pathogen secretes effector proteins to inhibit the immune response of the host plant using carbon sources in the soil and host plant cell walls.

[0004] Among them, glycoside hydrolases (GHs) are important secreted proteins of fungi, which can cleave the glycosidic bond of polysaccharide components. Their substrates are mainly cellulose, hemicellulose and pectin, etc., which can help fungal conidia to utilize polysaccharide components in the environment, help fungal spores to survive and complete infection. The spores of F. solani can survive in harsh conditions in the soil for a long time, which is an important source of initial infection. It has been reported that F. solani chlamydospores can colonize in fresh or rotten plant tissues and complete infection again. A large number of studies have shown that glycoside hydrolases play an important role in the pathogenicity of Fusarium oxysporum, Lagenidium giganteum and Magnaporthe oryzae. However, the research on glycoside hydrolases in F. solani is less.

[0005] Due to the ability of F. solani to survive in the soil for a long time and its wide host range, it is very difficult to control. Current research mainly focuses on the classification and identification of the pathogen and chemical pesticide control. So far, it is not clear whether the glycoside hydrolase coding gene FsGH28c has a function in the development, carbon utilization and pathogenicity of F. solani and whether it is related to pepper wilt. SUMMARY

[0006] Therefore, the present application aims to provide the application of glycoside hydrolase FsGH28c and its coding gene in regulating the pathogenicity of a bacterial strain.

[0007] The present application provides the application of at least one of the following ①~⑤ in regulating the pathogenicity of Fusarium solani.

[0008] ① glycoside hydrolase FsGH28c;

[0009] ② a protein having one or more amino acids substituted, deleted or added in the amino acid sequence of glycoside hydrolase FsGH28c and having the same or similar function as glycoside hydrolase FsGH28c;

[0010] ③ a nucleic acid molecule encoding the protein of ① or ②;

[0011] ④ a nucleic acid molecule having one or more nucleotides substituted, deleted or added in the nucleotide sequence of the nucleic acid molecule of ③ and being able to encode a protein having the same or similar function;

[0012] ⑤ a substance capable of regulating the level or activity of at least one of ①~④.

[0013] In some embodiments, the glycoside hydrolase FsGH28c has the amino acid sequence shown in SEQ ID NO: 1, and the coding gene of the glycoside hydrolase FsGH28c has the nucleotide sequence shown in SEQ ID NO: 2.

[0014] In some embodiments, the application further comprises regulating the carbon source utilization of Fusarium solani and / or regulating the growth and development of spores of Fusarium solani.

[0015] The present application provides a preparation for improving the pathogenicity, carbon source utilization rate and / or growth and development of spores of Fusarium solani, comprising at least one of the following (1)~(5):

[0016] (1) an expression vector for up-regulating the glycoside hydrolase FsGH28c coding gene;

[0017] (2) a recombinant host containing (1);

[0018] (3) a nucleic acid molecule for up-regulating the expression of the glycoside hydrolase FsGH28c coding gene;

[0019] (4) a strong promoter for the expression of the glycoside hydrolase FsGH28c coding gene;

[0020] (5) a preparation for promoting the transcription of the glycoside hydrolase FsGH28c coding gene;

[0021] (6) an activity promoter of a glycoside hydrolase FsGH28c-encoding gene.

[0022] The present application provides a preparation for reducing the pathogenicity, carbon source utilization rate, and / or spore growth and development of Fusarium solani f. sp. pisi, comprising at least one of the following 1) to 5):

[0023] 1) an expression vector for knocking out, knocking down, or silencing a glycoside hydrolase FsGH28c-encoding gene;

[0024] 2) a recombinant host containing (1);

[0025] 3) a nucleic acid molecule for down-regulating the expression of a glycoside hydrolase FsGH28c-encoding gene;

[0026] 4) a terminator or transposon for the expression of a glycoside hydrolase FsGH28c-encoding gene;

[0027] 5) a preparation for inhibiting the transcription of a glycoside hydrolase FsGH28c-encoding gene;

[0028] 6) an activity inhibitor of a glycoside hydrolase FsGH28c-encoding gene.

[0029] The present application provides the use of the preparation for increasing the pathogenicity, carbon source utilization rate, and / or spore growth and development of Fusarium solani f. sp. pisi or the preparation for reducing the pathogenicity, carbon source utilization rate, and / or spore growth and development of Fusarium solani f. sp. pisi in regulating the pathogenicity, carbon source utilization rate, and / or spore growth and development of Fusarium solani f. sp. pisi.

[0030] The present application provides a method for increasing the pathogenicity, carbon source utilization rate, and / or spore growth and development of Fusarium solani f. sp. pisi, using the preparation for increasing the pathogenicity, carbon source utilization rate, and / or spore growth and development of Fusarium solani f. sp. pisi, up-regulating the expression of a glycoside hydrolase FsGH28c-encoding gene, the transcription of a glycoside hydrolase FsGH28c-encoding gene, the level and / or activity of an endogenous glycoside hydrolase FsGH28c of a strain.

[0031] The present application provides a method for reducing the pathogenicity, carbon source utilization rate, spore growth and development, and / or preventing and treating pepper fusarium wilt, characterized by using the preparation for reducing the pathogenicity, carbon source utilization rate, and / or spore growth and development of Fusarium solani f. sp. pisi, knocking out, knocking down, or silencing the expression of a glycoside hydrolase FsGH28c-encoding gene of Fusarium solani f. sp. pisi, inhibiting the transcription of a glycoside hydrolase FsGH28c-encoding gene, reducing the level and / or activity of an endogenous glycoside hydrolase FsGH28c of a strain.

[0032] The present application provides a preparation for detecting pepper fusarium wilt, comprising:

[0033] an agent for detecting the transcription level of the glycoside hydrolase FsGH28c-encoding gene; and / or

[0034] an agent for detecting the expression level or activity of the glycoside hydrolase FsGH28c-encoding gene.

[0035] The present application provides a method for identifying pepper fusarium wilt, which detects the transcription level of the glycoside hydrolase FsGH28c-encoding gene of the fungus or detects the expression level or activity of the glycoside hydrolase FsGH28c-encoding gene by using the agent for detecting pepper fusarium wilt.

[0036] According to the specific embodiment of the present application, the single-gene mutant is obtained by knocking out or complementing the Fusarium solani pisi gene FsGH28c, and whether the knocking out or complementing mutant is successful is identified in the genome of the mutant by using PCR technology. Pepper is one of the host plants of Fusarium solani pisi, and plays an important role in indicating the strength of the pathogenicity of Fusarium solani pisi. In the method, the test of inoculating the wild-type strain and the FsGH28c-knocking out or complementing mutant into pepper can be performed by using the root-dipping method.

[0037] The present application has the following beneficial effects:

[0038] The present application provides a glycoside hydrolase FsGH28c-encoding gene related to the development, carbon source utilization and pathogenicity of Fusarium solani pisi. After knocking out the FsGH28c gene, the Fusarium solani pisi causes the decrease of sporulation, the abnormal development of spores, and the decrease of the utilization rate of carbon source (especially pectin). In addition, the amount of fungal colonization in the root of the host pepper and the pathogenicity of the FsGH28c gene deletion mutant are reduced. The FsGH28c gene of the present application positively regulates the pathogenicity of Fusarium solani pisi, and can be used as an important candidate gene for the study of the pathogenic mechanism of Fusarium solani pisi. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 A schematic diagram showing the knocking out and complementing of the FsGH28c gene of Fusarium solani pisi;

[0040] Figure 2 PCR verification results of the genome DNA of the successful mutant of the FsGH28c gene of Fusarium solani pisi;

[0041] Figure 3 PCR verification results of the genome DNA of the successful mutant of the FsGH28c gene of Fusarium solani pisi;

[0042] Figure 4 Changes in the mycelial development of the knocking out and complementing mutants, in which * represents p<0.05;

[0043] Figure 5The figure shows the changes in spore development between knockout and complement mutants. * represents p<0.05.

[0044] Figure 6 The figure shows the changes in carbon source utilization by knockout and replenishment mutants. * represents p<0.05, ** represents p<0.01, and *** represents p<0.001.

[0045] Figure 7 The disease incidence of susceptible pepper varieties inoculated with wild-type, knockout mutant, and complement mutants is shown.

[0046] Figure 8 The disease index of pepper varieties inoculated with wild-type, knockout mutant, and complement mutant is shown in the figure. * represents p<0.05.

[0047] Figure 9 This shows the isolation of fungi from the stems of pepper plants after inoculation;

[0048] Figure 10 The figure shows the analysis of fungal biomass in the stems of pepper plants after inoculation. ** represents p<0.01. Detailed Implementation

[0049] This invention provides the application of glycoside hydrolase FsGH28c and its encoding gene in regulating the pathogenicity of bacterial strains. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0050] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.

[0051] Example 1: Obtaining and identifying the FsGH28c knockout and complement mutant of Fusarium solani.

[0052] Using the highly pathogenic strain WN-1 of Fusarium rotundifolium as material, the upstream Up-1.0Kbp and downstream Down-1.0Kbp sequences of the FsGH28c gene were selected.

[0053] Information about the FsGH28c gene includes:

[0054] Gene Symbol: MRS44_007102

[0055] Old Locus Tag: B0J15DRAFT_426735

[0056] mRNA Accession: XM_046272235.2

[0057] Protein Accession: XP_046129366.1.

[0058] The CDS gene sequence of the Fusarium solani glycoside hydrolase encoding gene FsGH28c is as follows:

[0059]

[0060] The protein encoded by Fusarium solani pisi gene FsGH28c is a Glycoside Hydrolases, and its amino acid sequence is as follows:

[0061] MISSLFMLAGFAASALALPTSLEARAGCTFTDAASAIAGKGSCSTIILNNIAVPAGTTLDLTKLKDGTHVIFQGKTTFGYKEWEGPLIAFTGNNLLIEGASGHSIDCQGQRWWDTKGSNGGKTKPKFFSAHSLKNSNIKNLNVLNTPVQAFSINGVTNLGVYGVHLDNSLGDTQGGHNTDAFDVGSSNGVYISGAVVKNQDDCLAINSGTNITFTGGNCSGGHGLSIGSVGGRSDNVVKTVRILNSVIANSDNGVRIKTVYGATGSVSDVKYDTITLQNIAKYGIVIEQDYDNGSPKGTPSAGVPITDVTINKVVGTVKSSGTDVYILCASCKNWTWTGNSVTGGKKSDKCKGVPTGASC (SEQ ID NO: 1)

[0062] The following primer pairs B303-FsGH28c-UP-F / R and B303-FsGH28c-DOWN-F / R were used to amplify the wild type genomic DNA, and the primer information used for constructing the FsGH28c knockout, complementation vector and mutant PCR verification of Fusarium solani pisi is shown in Table 1.

[0063] Table 1, primer information used for constructing the FsGH28c knockout, complementation vector and mutant PCR verification of Fusarium solani pisi

[0064]

[0065] Homologous recombination technology was used to construct the FsGH28c gene upstream fragment Up-1.0 Kbp, downstream fragment Down-1.0 Kbp, and hygromycin fragment Hyg into the B303 vector, forming the FsGH28c knockout vector B303-Up-Hyg-Down. The B303-Up-Hyg-Down plasmid was transformed according to the manufacturer's instructions. Positive bacterial plasmid DNA was extracted according to standard chemical protocols. Then, the FsGH28c gene knockout mutant was obtained through protoplast genetic transformation of *Fusarium solani*. The construction method of the FsGH28c complementation vector was the same as that of the knockout vector. Similarly, in the context of the knockout mutant, complementary mutants were obtained through protoplast genetic transformation. Genomic DNA of the strains obtained from the above operations was extracted and verified using PCR technology.

[0066] like Figure 1 As shown, this illustrates the principle of fungal genome gene knockout. Using homologous substitution, the target gene FsGH28c in the *Fusarium solani* genome is replaced by the Hyg fragment in the vector B303-Up-Hyg-Down.

[0067] like Figure 2 As shown, among the eight knockout transformants obtained, transformant number 2 can detect the Hyg fragment but cannot detect the FsGH28c fragment, proving that transformant number 2 is a positive knockout transformant ΔFsGH28c.

[0068] like Figure 3 As shown, among the eight complement transformants obtained, transformant No. 2 can detect the FsGH28c fragment but cannot detect the Hyg fragment, proving that transformant No. 2 is a positive complement transformant C-ΔFsGH28c.

[0069] Example 2: Growth and development of knockout and addition mutants and wild-type mycelia.

[0070] Knockout, addition mutants, and wild-type strains were cultured on PDA medium and incubated in a 25°C incubator in the dark for 3 days. Then, 0.5 cm mycelial cakes were taken using a sterile punch and placed in the center of a new PDA medium. The mycelial cakes were incubated in a 25°C incubator in the dark for 7 days. The mycelial growth was measured using the cross-hatching method, and mycelia were picked and placed under a scanning electron microscope to observe mycelial development.

[0071] like Figure 4 As shown, compared with the complement mutant and the wild-type strain, the knockout mutant exhibited a significantly reduced hyphal growth rate, while hyphal phenotypic development showed no significant difference. This indicates that FsGH28c participates in regulating hyphal growth but does not affect the development of hyphal phenotypic characteristics.

[0072] Example 3: Growth and development of knockout and complement mutants and wild-type spores

[0073] The knockout, complementation mutant and wild type strains 0.5 cm bacterial cake were placed in PDB medium, 25°C constant temperature shaking table 180 rpm light culture for 3 days. Four layers of gauze filter, using blood cell counting plate to calculate the concentration of spore liquid in different strains of filtrate, and different strains of spores were placed under scanning electron microscope to observe the development of spores.

[0074] As shown in Figure 5 , compared with the complementation mutant (27.80 x 10 7 / mL) and wild type (28.60 x 10 7 / mL) strains, the spore amount of knockout mutant (16.94 x 10 7 / mL) was significantly reduced, the spore surface was invaginated, uneven and development was abnormal. It showed that FsGH28c was involved in the regulation of spore yield, and affected the phenotypic development of spores.

[0075] Example 4 Determination of the utilization efficiency of different carbon sources by knockout, complementation mutant and wild type strains

[0076] Solid Czapek's medium was added with sucrose (30 g / L), pectin (10 g / L), raffinose (10 g / L), cellulose (5 g / L) and galactose (10 g / L) respectively, for analyzing the utilization of different carbon sources by WT, ΔFsGH28c and C-ΔFsGH28c. The experiment was repeated 3 times.

[0077] As shown in Figure 6 , the growth rate of ΔFsGH28c strain in the medium with pectin as the only carbon source was the slowest, and the mycelial growth diameter was 1.31 cm after 7 days. It showed that the deletion of FsGH28c affected the utilization of pectin by Fusarium solani pisi.

[0078] Example 5 Pathogenicity identification of wild type, knockout mutant and complementation mutant strains

[0079] All strains (WT, ΔFsGH28c and C-ΔFsGH28c) were inoculated in PDB medium, 25°C constant temperature shaking table 180 rpm light culture for 3 days, four layers of gauze filter, then the spore suspension was diluted to 1 x 10 7 CFU / mL for subsequent experiments.

[0080] Pepper seedlings of variety "Reyin No. 1" were cultivated in vermiculite sand. After the seedlings developed four leaves, the roots were soaked in WT, ΔFsGH28c, and C-ΔFsGH28c spore suspensions for 15 minutes. After inoculation, the seedlings were transplanted into sterile nutrient soil. Disease surveys were conducted three times at 4, 6, and 8 weeks post-inoculation. Each strain was tested three times individually, with three replicates. The number of diseased plants was counted based on leaf disease incidence. Disease severity was classified into four levels: 0, 1 (less than 33% of leaves affected), 2 (more than 33% but less than 66% of leaves affected), 3 (more than 66% but less than 99% of leaves affected), and 4 (100%) for disease assessment. The assessment data were calculated using the disease index formula, as follows:

[0081] Disease index = [(0·n0 + 1·n1 + 2⋅n2 + 3⋅n3 + 4⋅n4) / 4⋅n] × 100%

[0082] Note: n0-n4 represent the number of plants for each disease level, and n represents the total number of plants surveyed each time.

[0083] like Figure 7 As shown, pepper seedlings inoculated with strain ΔFsGH28c experienced reduced leaf drop, a significant decrease in the number of diseased leaves, and a weakening of wilt symptoms.

[0084] like Figure 8 As shown, the results of the survey at 4, 6 and 8 weeks after inoculation of "Reyin No. 1" pepper seedlings with different mutant strains of Fusarium solani were as follows: the disease index of inoculation with the knockout mutant ΔFsGH28c was 21.66, 33.91 and 37.29, respectively; the disease index of inoculation with wild-type Fusarium solani was 31.93, 50.02 and 65.42, respectively; and the disease index of inoculation with complement mutant C-ΔFsGH28c was 30.88, 49.92 and 61.08, respectively.

[0085] 5.1 Isolation of fungi from pepper plant stems after inoculation

[0086] To investigate the fungal recovery of pepper plant stems inoculated with different strains, pepper stems were collected 8 weeks after inoculation. They were then disinfected by soaking in 3% NaClO, 75% ethanol, 50% ethanol, 25% ethanol, 20% ethanol, and sterile water, followed by rinsing. The stems were then aseptically cut into 1cm segments and placed on PDA plates. The plates were incubated in a 25°C incubator under darkness, and mycelial growth was observed after 3 days. The experiment was repeated three times.

[0087] like Figure 9 As shown, the number of stem segments from which pathogens were isolated from plants inoculated with strain ΔFsGH28c was significantly less than that from control plants.

[0088] 5.2 Fungal biomass detection

[0089] To compare the changes in pathogen biomass in pepper seedlings inoculated with different strains, this experiment used qRT-PCR to determine the biomass of *Fusarium solani* from different pepper stems. Total DNA was extracted from pepper stems 8 weeks after inoculation, and relative quantification was performed using the *Fusarium solani*-specific FsTEF gene as the target fragment and the pepper housekeeping gene PnMLF1 as the housekeeping fragment.

[0090] like Figure 10 As shown, the pathogen biomass in the stems of plants inoculated with strain ΔFsGH28c was significantly lower than that in the control plants.

[0091] Table 2. Primer Information for Quantitative Fluorescence Detection

[0092]

[0093] The FsGH28c gene of this invention plays an important positive regulatory role in the pathogenicity of Fusarium solani and can serve as an important candidate gene for studying the pathogenic mechanism of Fusarium solani. It provides new perspectives for further research on the pathogenic mechanism of Fusarium solani and increases the gene resources of pathogenicity-related genes of Fusarium solani.

[0094] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of glycoside hydrolase FsGH28c in reducing the pathogenicity of Fusarium solani: The amino acid sequence of the glycoside hydrolase FsGH28c is shown in SEQ ID NO:1, and the nucleotide sequence of the gene encoding the glycoside hydrolase FsGH28c is shown in SEQ ID NO:

2.

2. The application according to claim 1, characterized in that, The applications include reducing the carbon source utilization rate of Fusarium solani and / or inhibiting the growth and development of Fusarium solani spores.

3. A method for reducing the pathogenicity of Fusarium solani, characterized in that, Knockout, knockdown or silence the gene encoding glycoside hydrolase FsGH28c of Fusarium solani, inhibit the transcription of the gene encoding glycoside hydrolase FsGH28c of Fusarium solani, or reduce the expression level and / or activity of glycoside hydrolase FsGH28c of Fusarium solani. The amino acid sequence of the glycoside hydrolase FsGH28c is shown in SEQ ID NO:1, and the nucleotide sequence of the gene encoding the glycoside hydrolase FsGH28c is shown in SEQ ID NO:

2.

4. A method for controlling pepper wilt disease, characterized in that, Knockout, knockdown, or silence the gene encoding the glycoside hydrolase FsGH28c of Fusarium solani colonizing pepper, inhibit the transcription of the gene encoding the glycoside hydrolase FsGH28c of Fusarium solani colonizing pepper, or reduce the expression level and / or activity of the glycoside hydrolase FsGH28c of Fusarium solani colonizing pepper. The amino acid sequence of the glycoside hydrolase FsGH28c is shown in SEQ ID NO:1, and the nucleotide sequence of the gene encoding the glycoside hydrolase FsGH28c is shown in SEQ ID NO:2.

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