Use of a myxobacteria-derived beta-1,3-glucanase and compositions thereof in the biological control of phytophthora diseases

Abstract

This invention discloses the application of a β-1,3-glucanase derived from myxobacteria and its composition in the biocontrol of Phytophthora blight. This invention provides three β-1,3-glucanase genes that can be used for the biocontrol of Phytophthora blight, with nucleotide sequences of SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, and the amino acid sequences of the encoded glycoside hydrolase proteins of SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively. This β-1,3-glucanase and its composition can effectively inhibit the infection of plants by Phytophthora blight.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbiology and plant protection, and discloses a method for preparing three β-1,3-glucanases and their combinations from myxobacteria of the Sporangiaceae family, and their application in the prevention and control of Phytophthora blight in plants. Background Technology

[0002] Plant diseases are a major factor restricting high-quality and high-yield crops. It is estimated that disease losses in major crops worldwide account for approximately 20-40% of total yield, easily causing severe economic losses. Currently, plant diseases in agricultural production are mainly caused by bacteria, fungi, and oomycetes. Physical fumigation, chemical pesticide treatment, tillage and agricultural management, plant disease-resistant breeding, and biological control are effective means to suppress disease occurrence, playing a crucial role in efficient plant disease control and high-quality and high-yield crops. With the rapid development of gene editing technology, the shortening of plant disease-resistant breeding cycles makes the use of disease-resistant genes to improve plant disease resistance of significant research value. In this process, the discovery of high-performance antimicrobial proteins is crucial.

[0003] Fungal cell walls are primarily composed of polysaccharides such as β-1,3-glucan, β-1,6-glucan, and chitin. In contrast, the cell walls of oomycetes, represented by *Phytophthora*, are mainly composed of β-1,3-glucan and cellulose, with some members containing small amounts of β-1,6-glucan and chitin. Therefore, β-1,3-glucan is a conserved structure in the cell walls of plant-pathogenic eukaryotic microorganisms and an important target for the development of broad-spectrum antimicrobial drugs. For example, the plant-derived antimicrobial compound poacic acid inhibits the growth of pathogenic fungi by targeting the synthesis of β-1,3-glucan in their cell walls (JS Piotrowski et al., 2015). β-1,3-glucanases are an important class of antimicrobial agents and have been widely used in plant disease resistance breeding. Currently, β-1,3-glucanases with good antimicrobial activity are mainly derived from plants and microorganisms. However, reported β-1,3-glucanases vary considerably in activity and catalytic properties, and the complex cell wall structure of pathogenic bacteria is one of the important reasons for the significant differences in the antibacterial properties of β-1,3-glucanases. Furthermore, in the course of long-term evolution, pathogenic bacteria have resisted the hydrolysis of their cell walls by β-1,3-glucanases through dynamic changes in cell wall components and structural modifications, thereby weakening their antibacterial effects. Therefore, researchers have developed combinations of β-1,3-glucanase with proteases, and β-1,3-glucanase with chitinases, to enhance their antibacterial properties.

[0004] Myxobacteria are a novel class of biocontrol microorganisms with predatory characteristics, possessing significant application potential in plant disease control. Currently, broad-spectrum antimicrobial proteins derived from myxobacteria mainly include the application of β-1,6-glucanase from *Cordyceps militaris* in combating plant pathogenic fungi (ZL 201510816987.9), while patents on antimicrobial proteins such as β-1,3-glucanase from myxobacteria are relatively few. Based on the diversity of antagonistic strategies employed by myxobacteria against pathogens, such as inhibition and predation, this patent targets β-1,3-glucanase in the cell walls of eukaryotic microorganisms, represented by *Phytophthora infestans*, to develop β-1,3-glucanases and their compositions with broad-spectrum and highly efficient antifungal activity, which has significant application value for the biocontrol of *Phytophthora infestans*. Summary of the Invention

[0005] The purpose of this invention is to provide three novel β-1,3-glucanases derived from myxobacteria and their encoding genes. These enzymes can be identified by online BLAST alignment of amino acid sequence motifs (primary structure), secondary structural elements, and tertiary structural elements with the amino acid sequence. These β-1,3-glucanases originate from myxobacteria, belonging to the family Cystobacteraceae and the genus Archangium.

[0006] Another object of the present invention is to provide a genetically engineered bacterium containing the β-1,3-glucanase gene.

[0007] Another object of the present invention is to provide the application of the gene and the protein encoded by the gene.

[0008] The three β-1,3-glucanase genes acGlu13.1, acGlu13.2 and acGlu13.3 described in this invention have nucleotide sequences of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0009] The three β-1,3-glucanase genes described in this invention encode the β-1,3-glucanase proteins AcGlu13.1, AcGlu13.2, and AcGlu13.3, with amino acid sequences of SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively. The optimal reaction pH for β-1,3-glucanase AcGlu13.1 is 8.0, the optimal reaction temperature is 60℃, and its specific activity is 339.8 U / mg with laminarin as a substrate and 426.8 U / mg with poria cocos polysaccharide as a substrate. The optimal reaction pH for β-1,3-glucanase AcGlu13.2 is 7.0, the optimal reaction temperature is 60℃, its specific activity is 31.9 U / mg with laminarin as a substrate, and it has no activity against poria cocos polysaccharide. The optimal reaction pH for β-1,3-glucanase AcGlu13.3 is 6.0, the optimal reaction temperature is 60℃, the specific activity is 757.3 U / mg with kelp polysaccharide as substrate, and the specific activity is 154.4 U / mg with poria polysaccharide as substrate.

[0010] This invention provides online BLAST sequence alignment of β-1,3-glucanase amino acid sequences, obtaining sequences from the Cynotrophae family with more than 50% homology to the β-1,3-glucanases of this invention. The proteins encoded by the obtained homologous sequences are from the NCBI database, and their functions have not been verified. This invention randomly selected representative proteins with more than 50% homology to the three β-1,3-glucanases described in this invention, and from five genera within the Cynotrophae family (Archangium, Stigmatella, Cystobacter, Hyalangium, and Vitiosangium), for heterologous expression function verification. It was found that homologous proteins from different sources all possess β-1,3-glucan hydrolytic activity. These proteins... The NCBI accession numbers are WP_204490482.1, EAU62082.1, WP_095981735.1, WP_108065359.1, EPX59331.1, WP_044196135.1, WP_239470089, ADO71648.1, WP_020918155, WP_224360605, and WP_075207634EPX62475.1, etc. The functions of these proteins also fall within the scope of protection of this invention.

[0011] Recombinant microorganisms containing the β-1,3-glucanase gene described in this invention are preferably Escherichia coli as the host bacterium.

[0012] The present invention provides an enzyme composition comprising the β-1,3-glucanase and its homologous proteins described in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, any two or three of which are combined. To enhance the antibacterial effect, preferably, the concentrations of AcGlu13.1, AcGlu13.2 and AcGlu13.3 are ≥0.1 μg / mL.

[0013] The β-1,3-glucanase and its enzyme composition described in this invention are preferably used in the prevention and control of damage to plants caused by Phytophthora.

[0014] Among them, the diseases caused by the plant pathogenic oomycete Phytophthora include: Potato Late Blight, Soybean Phytophthora, Pepper Phytophthora, Tobacco Phytophthora, etc.

[0015] Beneficial effects

[0016] 1. This invention uses the myxobacterial strain *Protozoa* screened from soil samples as material. Through purification and identification of fermentation supernatant protein, three highly active β-1,3-glucanases, AcGlu13.1, AcGlu13.2, and AcGlu13.3, were obtained, with specific activities of 339.8, 31.9, and 757.3 U / mg, respectively, using laminarin as a substrate. The β-1,3-glucanase gene sequences were successfully obtained through protein amino acid sequencing combined with PCR amplification, and named acGlu13.1, acGlu13.2, and acGlu13.3, respectively.

[0017] 2. Sequence comparison revealed protein sequences with over 50% homology to AcGlu13.1, AcGlu13.2, and AcGlu13.3. It was found that the three proteins described in this patent and their homologous proteins all originate from five genera within the Cynocystaceae family: Archangium, Stigmatella, Cystobacter, Hyalangium, and Vitiosangium. Furthermore, gene synthesis and heterologous expression of related homologous proteins were performed to verify their function, revealing that these homologous proteins all possess β-1,3-glucan hydrolytic activity.

[0018] 3. Using the three β-1,3-glucanases described in this patent, by combining them in pairs or in groups of three, it was found that compositions based on two or three enzymes significantly increased the antagonistic activity against Phytophthora indices at concentrations of ≥0.1 μg / mL.

[0019] 4. The fermentation supernatant of Protozoa AC19 containing β-1,3-glucanase significantly inhibited the growth of Phytophthora soybeanis and had a good biocontrol effect on Phytophthora disease.

[0020] 5. Mixtures containing this β-1,3-glucanase and their transgenic plant materials can be used for plant health protection, agricultural disease control, and agricultural product processing. Attached Figure Description

[0021] Figure 1 SDS-PAGE electrophoresis images of fermentation supernatant and purified protein adsorbed from cell walls of myxobacterium AC19. Lane 1: Protein marker; Lane 2: Extracted cell wall; Lane 3: Fermentation supernatant; Lane 4: Fermentation supernatant after ultrafiltration; Lane 5: Protein adsorbed from cell walls.

[0022] Figure 2 SDS-PAGE electrophoresis images of recombinant β-1,3-glucanases AcGlu13.1, AcGlu13.2, and AcGlu13.3.

[0023] Figure 3 Enzymatic properties of recombinant β-1,3-glucanases AcGlu13.1, AcGlu13.2, and AcGlu13.3: a, c, e: Effects of temperature on the enzyme activities of AcGlu13.1, AcGlu13.2, and AcGlu13.3, respectively; b, d, f: Effects of pH on the enzyme activities of AcGlu13.1, AcGlu13.2, and AcGlu13.3, respectively.

[0024] Figure 4 Effects of recombinant β-1,3-glucanase and its composition on the growth of Phytophthora in different protein concentrations

[0025] Figure 5 Biocontrol Effects of Recombinant β-1,3-glucanase and its Composition on Soybean Phytophthora blight

[0026] a. Phenotypic features of hypocotyl disease in soybean yellowing seedlings; b. Statistical analysis of hypocotyl lesion length in different treatment groups.

[0027] Information on the preservation of biological materials

[0028] Archangium sp. AC19 is deposited at the Guangdong Provincial Microbial Culture Collection Center, located at the Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, China. The deposit date is January 13, 2022, and the accession number is GDMCC No: 62205. Detailed Implementation

[0029] Example 1: Purification and gene cloning of β-1,3-glucanase

[0030] Strain Archangium sp. AC19 (GDMCC No: 62205) was inoculated into VY / 4 liquid medium (VY / 4 medium: 0.25% yeast cells, 0.1% CaCl2, pH 7.0) and cultured in a shaker at 30°C for 2-3 days. The fermentation supernatant was collected by centrifugation, and concentrated using an 80% ammonium sulfate gradient precipitation. The supernatant was then purified using the Phytophthora cell wall as an affinity matrix to obtain candidate proteins with potential substrate-binding capabilities (such as...). Figure 1 Three candidate β-1,3-glucanases were screened and obtained through mass spectrometry identification combined with subsequent functional verification. Based on the genome sequence of the myxobacterium Archangium sp. AC19 and combined with NCBI genomic information for ORF prediction, PCR amplification of the β-1,3-glucanase gene was performed using the genomic DNA of Protozoa AC19 (GDMCC No: 62205) as a template to obtain the full-length sequence of the target gene. The AcGlu13.1 gene has a full length (from start codon to stop codon) of 2376 bp, and its sequence is SEQ ID NO.1, encoding 791 amino acids. Its amino acid sequence is SEQ ID NO.4, and the first 28 amino acids at the N-terminus of this protein are a signal peptide. The AcGlu13.2 gene has a full length (from start codon to stop codon) of 1911 bp, and its sequence is SEQ ID NO.2, encoding 636 amino acids. Its amino acid sequence is SEQ ID NO.5, and the first 32 amino acids at the N-terminus of this protein are a signal peptide. The AcGlu13.3 gene has a full length (from start codon to stop codon) of 1287 bp, and its sequence is SEQ ID NO.3, encoding 429 amino acids. Its amino acid sequence is SEQ ID NO.6, and the first 12 amino acids at the N-terminus of this protein are a signal peptide. The primers used for expression of AcGlu13.1 in E. coli were F1 and R1, the primers used for expression of AcGlu13.2 in E. coli were F2 and R2, and the primers used for expression of AcGlu13.3 in E. coli were F3 and R3. The amplification sequence used for heterologous expression in E. coli did not contain the signal peptide sequence of the protein.

[0031] F1: 5-GCtctagaATGGCGACGCCCATTTCCCAG-3(Xba I);

[0032] R1: 5-CCCAAGCTTTCAGTGGTGGTGGTGGTGGTGCTTGGTCCAGGTGTA-3 (Hind III)

[0033] F2: 5-GCtctagaATGGCGCCGAGCGCGCAGGTCAT-3(Xba I);

[0034] R2: 5-CCCAAGCTTTCAGTGGTGGTGGTGGTGGTGCTGCTTGACGAGCTCGAA-3 (Hind III)

[0035] F3: 5-GCtctagaATGGCGTGCGCGCCCGAGGCCT-3(Xba I);

[0036] R3: 5-CCCAAGCTTTCAGTGGTGGTGGTGGTGGTGCTGCCACTGGTAGACGCGCA-3(Hind III)

[0037] Example 2: Expression and purification of β-1,3-glucanase in Escherichia coli

[0038] Using *E. coli* BL21 as the expression host, the PCR amplification product of the β-1,3-glucanase encoding gene (with the signal peptide removed) was ligated into the pET29a vector via enzyme ligation to construct plasmids pET29a-acGlu13.1, pET29a-acGlu13.2, and pET29a-acGlu13.3. These plasmids were then introduced into *E. coli* BL21(DE3) competent cells, plated on LB agar plates containing 50 mg / L kanamycin, and single colonies were picked and sequenced to confirm the presence of *E. coli* cells containing the pET29a-acGlu13.1, pET29a-acGlu13.2, and pET29a-acGlu13.3 plasmids. The constructed *E. coli* cells containing the expression plasmids were induced to express the recombinant protein using IPTG. The recombinant protein was purified using Ni column affinity chromatography. The results showed that ( Figure 2 The β-1,3-glucanase was well expressed in Escherichia coli. High-purity heterologous recombinant β-1,3-glucanase was obtained by Ni column purification. SDS-PAGE results showed that all three β-1,3-glucanases could be purified by Ni column to obtain high-purity recombinant proteins, and the electrophoretic molecular weight was basically consistent with their theoretical molecular weight.

[0039] Example 3: Study on the enzymatic properties of β-1,3-glucanase

[0040] The enzymatic properties of the obtained recombinant protein were studied, and the optimal reaction temperature was determined: the activity of the obtained recombinant protein was measured under different temperatures (30℃, 40℃, 50℃, 55℃, 60℃, 70℃, 80℃) and pH 6.0-8.0 conditions, and the highest enzyme activity was set as 100%. Figure 3a, c, e). Determination of optimal reaction pH: The activity of the purified recombinant protein was measured at 60℃ at different pH values ​​(3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0), with the highest activity set at 100%. Figure 3 (b, d, e). The results showed that the optimal temperature for AcGlu13.1 was 60℃ and the optimal pH was PBS buffer (pH 8.0); the optimal temperature for AcGlu13.2 was 60℃ and the optimal pH was Tris-HCl (pH 7.0); and the optimal temperature for AcGlu13.3 was 60℃ and the optimal pH was PBS buffer (pH 6.0).

[0041] Example 4: β-1,3-glucanase sequence analysis and functional verification of homologous proteins

[0042] NCBI online BLAST alignment of the β-1,3-glucanases AcGlu13.1, AcGlu13.2, and AcGlu13.3 sequences showed that AcGlu13.1 was predicted as a hypothetical protein, while AcGlu13.2 and AcGlu13.3 were predicted as glycoside hydrolases; their specific β-1,3-glucan hydrolase functions are unknown. Based on reported myxobacterial whole-genome sequences, the protein sequences of AcGlu13.1, AcGlu13.2, and AcGlu13.3 were analyzed using an online BLAST server and the ClustalW program (http: / / www.ebi.ac.uk / Tools / msa / clustalw2 / ). The results showed that AcGlu13.1, AcGlu13.2, and their homologs mainly originated from the Sporobacteriaceae family, while AcGlu13.3 and its homologs originated from the Myxococciaceae and Sporobacteriaceae families. Expression vectors were constructed using the coding genes of representative homologous proteins, and the β-1,3-glucanase activity of the recombinant proteins after induced expression was measured, following the procedures outlined in Examples 1 and 2. The results showed that, using laminarin as a substrate, homologous proteins derived from Myxococci and Sporobacteriaceae families all exhibited β-1,3-glucanase activity (Table 1). The above results illustrate the following: 1) These functional proteins from different strains have a sequence identity of more than 50% with the β-1,3-glucanases AcGlu13.1, AcGlu13.2, and AcGlu13.3 described in this patent; 2) As verified by Example 4 of this patent (Table 1), these functional proteins from different strains all have the same β-1,3-glucan hydrolase function as the proteins AcGlu13.1, AcGlu13.2, and AcGlu13.3 described in this patent. Therefore, it can be proven that these functional proteins from different strains are homologous proteins of AcGlu13.1, AcGlu13.2, and AcGlu13.3 described in this patent and should fall within the protection scope of this patent.

[0043] Table 1 Functional validation of AcGlu13.1, AcGlu13.2 and AcGlu13.3 homologous proteins

[0044] strain Protein and sequence number Sequence identical to GluM protein OD540 Archangium sp.AC19 AcGlu13.1 100％ 1.32 Archangium primigenium WP_204490482.1 78％ 0.91 Stigmatella aurantiaca EAU62082.1 70％ 0.65 Melittangium boletus WP_095981735.1 81％ 0.58 Vitiosangium WP_108065359.1 90％ 1.42 Cystobacter fuscu EPX59331.1 74％ 0.64 Hyalangium minutum WP_044196135.1 75％ 0.55 Archangium sp.AC19 AcGlu13.2 100％ 0.69 Archangium violaceum WP_239470089 94％ 0.57 Stigmatella aurantiaca ADO71648.1 84％ 0.42 Cystobacter fuscus WP_020918155 84％ 0.55 Hyalangium WP_224360605 73％ 0.79 Archangium sp.AC19 AcGlu13.3 100％ 1.59 Archangium violaceum WP_075207634 71％ 1.11 Cystobacter fuscus EPX62475.1 73％ 0.72

[0045] Example 5: Study on the antibacterial properties of β-1,3-glucanase and its composition

[0046] Using Phytophthora sojae P6497 as the model strain, the antibacterial effects of β-1,3-glucanases AcGlu13.1, AcGlu13.2, and AcGlu13.3 and their different combinations against Phytophthora P6497 were determined by 96-well plate experiments. Phytophthora P6497 was inoculated onto V8 solid medium and cultured at 25°C for 3-4 days. Healthy hyphae with vigorous margins were selected, and agar blocks of the same size were transferred to 96-well plates containing V8 liquid medium. Single enzymes (AcGlu13.1, AcGlu13.2, and AcGlu13.3 protein concentrations of 0.1, 0.5, 1, and 5 μg / mL, respectively) were added, along with pairwise combinations (AcGlu13.1 + AcGlu13.2; AcGlu13.2 + AcGlu13.3; AcGlu13.1 + AcGlu13.3, with final protein concentrations of 0.2, 1, 2, and 10 μg / mL) and three-enzyme combinations (AcGlu13.1 + AcGlu13.2 + AcGlu13.3, with final protein concentrations of 0.3, 1.5, 3, and 15 μg / mL). The buffer used was 50 mM Tris-HCl (pH 7.0). Incubate at 25℃ for 24-36 hours, observe mycelial growth, and quantitatively measure mycelial biomass. Results are as follows: Figure 5 Different enzyme concentrations inhibited the growth of *Phytophthora indicum* to varying degrees. Single enzyme treatments showed weak inhibitory effects at concentrations of 0.1 μg / mL and 0.5 μg / mL. However, pairwise combinations (AcGlu13.1 + AcGlu13.2; AcGlu13.2 + AcGlu13.3; AcGlu13.1 + AcGlu13.3) and three-enzyme combinations (AcGlu13.1 + AcGlu13.2 + AcGlu13.3) significantly inhibited the growth of *Phytophthora indicum*. Figure 5 The above results indicate that β-1,3-glucanases AcGlu13.1, AcGlu13.2, and AcGlu13.3 have a synergistic effect in antagonizing antifungal fungi, and that the two-enzyme combination and the three-enzyme combination have better antagonistic effects than the single enzyme.

[0047] Example 6: Evaluation of the biocontrol effect of β-1,3-glucanase and its composition on Phytophthora blight

[0048] Soybeans of the Williams variety susceptible to the disease were sown in vermiculite and cultured in the dark in a greenhouse for three days. Afterward, etiolated seedlings were removed from the soil and fixed onto inoculation trays. *Phytophthora solanacea* P6497 cultured in Example 5 was selected, and agar blocks of the same size were cut and transferred to an enzyme solution containing a single enzyme (0.01 mg / mL) and a three-enzyme combination (0.03 mg / mL). The buffer solution used was 50 mM Tris-HCl (pH 7.0), and the reaction temperature and time were 30℃ and 30 min, respectively. A buffer treatment was used as a control. The treated agar blocks were washed with buffer solution and then transferred to the hypocotyl of the etiolated seedlings. The seedlings were then kept moist with plastic wrap and cultured for 36 hours. Infection results were observed, and lesion lengths were counted. Results are as follows: Figure 5 As shown in a, single enzyme treatments of AcGlu13.1, AcGlu13.2, and AcGlu13.3 at a concentration of 0.01 mg / mL significantly reduced the infection of soybeans by Phytophthora infestans, and the lesions were significantly smaller. Meanwhile, the combined treatment group of the three enzymes showed even smaller lesion diameters, demonstrating a significant synergistic effect compared to single enzyme treatments (5b).

Claims

1. A β-1,3-glucanase gene, characterized in that, The gene is selected from either SEQ ID NO.2 or SEQ ID NO.

3.

2. The β-1,3-glucanase encoded by the β-1,3-glucanase gene of claim 1, characterized in that The amino acid sequences are SEQ ID NO.5 and SEQ ID NO.6, respectively.

3. A recombinant expression vector containing the β-1,3-glucanase gene as described in claim 1.

4. A genetically engineered bacterium containing the recombinant expression vector of claim 3, or a genetically engineered bacterium obtained by direct transformation or transduction of a host bacterium using the β-1,3-glucanase gene of claim 1, characterized in that... With E. coli BL21(DE3) as the host bacteria.

5. The application of the genetically engineered bacteria according to claim 4 for fermentation to produce β-1,3-glucanase.

6. Use of a β-1,3-glucanase for the biological control of Phytophthora sojae, characterized in that, The β-1,3-glucanase is selected from the β-1,3-glucanase shown in SEQ ID NO.5 or SEQ ID NO.

6.

7. The application of a β-1,3-glucanase composition in the biological control of soybean Phytophthora, characterized in that, The composition is selected from any two or three of the β-1,3-glucanases described in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.

6.

8. The use of the β-1,3-glucanase of claim 6 or the composition of claim 7 in improving the resistance of plants to Phytophthora soybeanis.

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