Application of comQ gene of bacillus subtilis and comQ gene knockout mutant of bacillus subtilis in improving resistance of plant induction system

Abstract

The invention discloses application of a bacillus subtilis comQ gene and a bacillus subtilis comQ gene knockout mutant in improving the resistance of a plant induction system. The mutant is obtained through directed mutation, and compared with wild type bacillus subtilis 168, the mutant shows remarkable induction resistance ability to rice. Experimental data show that after the delta comQ mutant strain is treated, the rice shows obvious sheath blight resistance, the ROS level and the disease-resistant related gene level of the rice root are remarkably improved, and the scab area is remarkably reduced. The invention provides a new thought for the development of microbial pesticides, and is beneficial to promoting the high-quality development of green agriculture. Meanwhile, the application value of the bacillus in agricultural production can be improved, and the effect of improving the farmland ecological environment is expected to be achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological control. Specifically, the present invention relates to the application of Bacillus subtilis comQ gene and Bacillus subtilis comQ gene knockout mutants in enhancing plant induced systemic resistance. Background Art

[0002] Rice, as the staple food that sustains more than half of the world's population, its yield and quality play a decisive role in food security. Rice sheath blight, belonging to the three major rice diseases, is a typical soil-borne disease caused by Rhizoctonia solani. During the whole growth period from seedling stage to ear stage of rice, it is threatened. The pathogen spreads among soil, field weeds and seedlings through irrigation water by means of sclerotia and mycelia. The infection route starts from the leaf sheath and gradually spreads to key parts such as leaves, stems and panicle necks, resulting in yellowing and death of leaves, seriously interfering with the normal growth of rice.

[0003] In recent years, the occurrence of rice sheath blight in rice-growing areas of China has shown an explosive growth trend. As a result, phenomena such as lodging and increased empty and shriveled grains have occurred in rice, and both the yield and quality have suffered serious impacts, causing huge economic losses.

[0004] For a long time, chemical control has dominated the control of rice sheath blight. However, according to the monitoring by relevant departments in recent years, there have been rice sheath blight pathogens resistant to chemical agents in many areas, and their population is large and widely distributed. Due to the lack of immune or highly resistant rice varieties at present, the control of sheath blight has become increasingly difficult, and a multi-faceted prevention and control strategy is urgently needed.

[0005] Biological control, as an environmentally friendly control strategy, is becoming increasingly important. Biocontrol bacteria play a key role in biological control. Their unique mechanism of inducing plant systemic resistance provides a new direction for the control of diseases such as rice sheath blight.

[0006] Induced systemic resistance (ISR) is an important part of the plant defense mechanism and can enable plants to have broad-spectrum resistance to a variety of pathogens. Compared with the basic resistance of plants themselves, ISR is more efficient and persistent, and can help plants resist a variety of diseases in a complex and changeable ecological environment. Biocontrol bacteria are efficient elicitors of ISR. By interacting with plants, they activate the plant's own immune signaling pathway, prompting the plant to produce a series of physiological and biochemical reactions, enhancing the plant's resistance to pathogens. ISR can not only help plants resist the invasion of current pathogens, but also provide long-term protection for the future growth of plants, greatly reducing the occurrence frequency and harm degree of diseases.

[0007] Among many biocontrol bacteria, Bacillus subtilis has been widely used in the control of plant diseases due to its unique advantages. Bacillus subtilis can colonize around plant roots, form endospores, have strong stress resistance, and have antagonistic effects against a variety of plant pathogenic fungi. At the same time, it is easy to store and use, has good stability when mixed with chemical pesticides, is non-toxic to humans and animals, and does not pollute the environment.

[0008] The ComQXPA quorum sensing system of Bacillus subtilis plays a core role in its interaction with plants. Quorum sensing (QS) is a mechanism for microorganisms to communicate with each other relying on chemical signals. The ComQXPA system consists of genes such as comA, comP, comQ, and comX. Among them, the ComQ isoprenyltransferase encoded by the comQ gene is responsible for recognizing and processing the precursor signal molecule pre-ComX, cleaving it into the biologically active ComX signal peptide, and then secreting it extracellularly. The ComX signal peptide then binds to the histidine kinase ComP on the cell membrane, activates the downstream signal pathway, regulates multiple physiological activities of the bacteria, and affects the interaction process between the bacteria and plants.

[0009] Although Bacillus subtilis has been widely used in the control of plant diseases, the physiological mechanism of its resistance to rice, especially the role of the comQ gene in the ComQXPA quorum sensing system in the communication process between Bacillus subtilis and rice, still needs to be further explored. In-depth study of the role of this gene in inducing systemic resistance in rice will not only help to reveal the complex molecular interaction mechanism between microorganisms and plants and provide support for the development of biocontrol theory, but also provide ideas for developing new, efficient, and green control strategies for rice sheath blight, which is of extremely important significance for ensuring the safe production of rice and promoting the sustainable development of green agriculture. Summary of the Invention

[0010] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies existing in the prior art, and to provide an application of the comQ gene of the ComQXPA quorum sensing system of Bacillus subtilis in improving the ability of Bacillus subtilis to induce induced systemic resistance in plants.

[0011] The second purpose of the present invention is to provide an application of the Bacillus subtilis ΔcomQ (comQ gene knockout) mutant strain in enhancing plant induced systemic resistance.

[0012] The third purpose of the present invention is to provide a method for enhancing plant induced systemic resistance to control plant diseases.

[0013] The above objects of the present invention are achieved by the following technical solutions:

[0014] The present invention uses the method of co-culturing by adjacent inoculation, with Bacillus subtilis as the research material. It is found that Bacillus subtilis 168 and its comQ gene deletion mutant have no antagonistic or competitive effect on Rhizoctonia solani Rs7. However, after treating with Bacillus subtilis for 1 day, by detecting the disease-resistant related genes in rice leaves and roots through Real-Time quantitative PCR (RT-qPCR), it is found that the treatment with Bacillus subtilis can activate the expression of disease-resistant related genes through the salicylic acid (SA) pathway, and at the same time, the ROS level in the rice roots also increases significantly. Among them, the ΔcomQ mutant is the most significant in enhancing the ability of rice to resist sheath blight. After treating rice leaves with Bacillus subtilis 168 and its ΔcomQ mutant and then inoculating with Rhizoctonia solani Rs7, transcriptome sequencing (RNA-seq) analysis is carried out, and it is found that Bacillus subtilis and its ΔcomQ mutant jointly regulate and promote the synthesis of PR1 protein through the Mitogen-Activated Protein Kinase (MAPK) pathway, the salicylic acid disease-resistant pathway, and the glutathione metabolism pathway, enabling rice to have the ability of induced systemic resistance.

[0015] Therefore, the present invention provides the application of the comQ gene of the ComQXPA quorum sensing system of Bacillus subtilis in improving the ability of Bacillus subtilis to induce induced systemic resistance in plants.

[0016] Furthermore, the application is to inhibit the expression of the comQ gene in Bacillus subtilis or knockout the comQ gene in Bacillus subtilis.

[0017] Furthermore, the comQ gene regulates the biocontrol mechanism of Bacillus subtilis, further affecting the disease resistance and immunity of plants.

[0018] The present invention also provides the application of the Bacillus subtilis ΔcomQ mutant strain in enhancing the induced systemic resistance of plants.

[0019] Furthermore, the Bacillus subtilis ΔcomQ mutant strain is the Bacillus subtilis 168ΔcomQ mutant strain.

[0020] Furthermore, the construction method of the Bacillus subtilis 168ΔcomQ mutant strain is the homologous recombination method.

[0021] Furthermore, the enhancement of the induced systemic resistance of plants is manifested as increasing the expression level of plant disease-resistant genes, the level of reactive oxygen species (ROS) in plants, and / or reducing the lesion area of plant leaves.

[0022] The present invention also provides a method for enhancing plant induced systemic resistance to control plant diseases, which is to apply a fermentation culture broth bacterial suspension of Bacillus subtilis ΔcomQ mutant strain to plants.

[0023] The specific method is as follows: The activated Bacillus subtilis 168ΔcomQ mutant strain is resuspended with rice nutrient solution until the OD value of the bacterial solution reaches about 1.0. 600 Subsequently, the obtained bacterial suspension is added to the rice nutrient solution. When adding, it is accurately calculated according to the volume of the nutrient solution to ensure that the OD value of the whole nutrient solution is finally adjusted to 0.001 after adding the bacterial suspension. 600 Value is finally adjusted to 0.001.

[0024] Furthermore, the application method is spraying, root irrigation or root dipping.

[0025] Furthermore, the plant is rice.

[0026] Furthermore, the plant disease is rice sheath blight.

[0027] The present invention also provides the application of Bacillus ΔcomQ mutant strain and comQ gene in preparing plants with high disease resistance and immunity.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention provides the application of Bacillus subtilis comQ gene and Bacillus subtilis comQ gene knockout (ΔcomQ) mutant in enhancing plant induced systemic resistance. By using the adjacent inoculation co-culture method, it is found that Bacillus subtilis 168 and its mutant ΔcomQ have no antagonistic or competitive effect on Rhizoctonia solani Rs7. However, one day after treatment with Bacillus subtilis 168 and its ΔcomQ mutant, the disease-resistant marker genes in rice leaves and roots are detected by RT-qPCR. It is found that the treatment with Bacillus subtilis up-regulates the expression of rice disease-resistant related genes and simultaneously increases the ROS level in rice roots. Among them, the ΔcomQ mutant has the most significant effect on the changes in the expression of rice resistance genes and the ROS level. At this time, Rhizoctonia solani Rs7 is inoculated on the rice leaves, and it is found that the rice leaves treated with Bacillus have the effect of resisting sheath blight, and the disease-resistant ability of the rice in the ΔcomQ mutant treatment group is improved most significantly. Transcriptome sequencing analysis is carried out on the rice leaves treated with Bacillus subtilis 168 and its ΔcomQ mutant and inoculated with Rhizoctonia solani Rs7. It is found that ΔcomQ can promote the synthesis of pathogenesis-related protein 1 (PR1) and the production of ROS through the synergistic effect of the MAPK signaling pathway and the salicylic acid-mediated plant disease-resistant signaling pathway, enabling rice to exert immune disease resistance. The activation of the glutathione pathway can enhance the antioxidant capacity of plants, maintain the redox balance in cells, enable plants to better maintain the normal functions and structures of cells when attacked by pathogens, and thus enhance the resistance to diseases, that is, the comQ gene can improve the ability of Bacillus subtilis to induce plant induced systemic resistance, and the ΔcomQ mutant strain can enhance plant induced systemic resistance. The present invention provides a new idea for the development of microbial pesticides, which helps to promote the high-quality development of green agriculture. At the same time, the invention can improve the application value of Bacillus in agricultural production and is expected to improve the farmland ecological environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Adjacent co-culture of Bacillus subtilis and Rhizoctonia solani Rs7. Note: Bs represents Bacillus subtilis; the green * represents the inoculation site of Bacillus; the red * represents the inoculation site of Rs7; the red dotted line indicates the growth range of Rs7

[0031] Figure 2 Effect of treatment with Bacillus subtilis on the expression of genes related to induced resistance in rice roots.

[0032] Figure 3 Effect of treatment with Bacillus subtilis on the expression of genes related to induced resistance in rice leaves.

[0033] Figure 4 Detection result of ROS in the root cap of rice roots treated with Bacillus subtilis.

[0034] Figure 5 ROS detection results of the elongation zone of rice roots treated with Bacillus subtilis.

[0035] Figure 6 Induction of sheath blight resistance in detached rice by Bacillus subtilis.

[0036] Figure 7 Induction of sheath blight resistance in living rice by Bacillus subtilis.

[0037] Figure 8 Analysis of the results of rice transcriptome sequencing (RNA-seq) under the treatment of Bacillus subtilis. Note: Green represents activation, and the red box represents significant upregulation. Detailed implementation mode

[0038] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0039] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0040] The Bacillus subtilis 168 strain was purchased from the Guangdong Provincial Microbial Culture Collection Center, and the strain preservation number is GDMCC NO.: 1.258.

[0041] The method for constructing the mutant ΔcomQ is homologous recombination.

[0042] Rhizoctonia solani 7 (Rs7) was purchased from the Guangdong Provincial Microbial Culture Collection Center, and the strain preservation number is GDMCC NO.: 3.700 (other number: CGMCC 3.7376).

[0043] Example 1 Co-culture of Bacillus and the pathogen of rice sheath blight

[0044] Co-culture method of adjacent inoculation of Bacillus and the pathogen of rice sheath blight (Rhizoctonia solani Rs7):

[0045] Use a punch with a diameter of 0.5 cm to punch holes in the culture plate of Rhizoctonia solani Rs7, and take the agar block at the edge of the colony and inoculate it in the center of the PDA plate. Pick the Bacillus subtilis 168 and the mutant ΔcomQ from the plate and inoculate them into 50 mL of LB liquid medium, and culture at 160 rpm and 30 °C for 4 - 5 hours until the OD of the bacterial liquid 600 is 0.4. Then, dilute the bacterial liquid with fresh liquid LB in proportion to the initial OD 600 to 0.004, and continue to culture until the OD 600It was 0.4. 1 μL of the bacterial solution was aspirated and added dropwise onto a PDA plate 1 cm away from the pathogenic fungal agar block for co-culture, and the interaction between the Bacillus and the pathogenic fungus was observed.

[0046] The results of the co-culture experiment of Bacillus and Rhizoctonia solani Rs7 are as Figure 1 shown. Compared with the control (CK) group, Bacillus subtilis 168 (Bs168) could, to a certain extent, hinder the growth of Rhizoctonia solani Rs7, slowing down its growth rate, but the hyphae of Rs7 could still extend and cover part of the colony of Bacillus subtilis 168. In addition, the Rs7 hyphae could completely cover the colony of the ΔcomQ mutant. This indicates that neither Bacillus subtilis 168 nor the ΔcomQ mutant could inhibit the growth of Rhizoctonia solani Rs7.

[0047] Example 2 Effects of Bacillus treatment on the disease resistance of rice

[0048] 1. Effects of Bacillus treatment on the expression of rice-induced resistance-related genes

[0049] Single colonies of Bacillus (Bacillus subtilis 168 and its ΔcomQ mutant) were picked from the plate and cultured in 50 mL of LB at 160 rpm and 30 °C for 4 - 5 hours until the OD of each bacterial solution 600 was all 0.4. Then, the bacterial solution was diluted proportionally with LB to an initial OD 600 of 0.004 and continued to be cultured until the OD 600 was 0.4. Centrifuged at 5000 rpm for 10 min at 4 °C, the supernatant was discarded, resuspended with sterile water, and then centrifuged at 5000 rpm for 10 min at 4 °C again, and resuspended with rice nutrient solution until the OD 600 was about 1.0. Subsequently, the obtained bacterial suspension was added to the rice nutrient solution. When adding, it was accurately calculated according to the volume of the nutrient solution to ensure that after adding the bacterial suspension, the OD 600 value of the whole nutrient solution was finally adjusted to 0.001. The rice was soaked in the nutrient solution and cultured under the conditions of 25 °C, 75% humidity, 12-hour light, and 12-hour darkness. After 24 hours of treatment, samples were taken from the roots and leaves of the rice. The RNA of the samples was extracted, then reverse transcribed, and the transcriptional levels of disease resistance-related genes in the rice leaves and roots were detected by RT-qPCR, and the relative quantitative analysis of the gene expression levels was performed.

[0050] The quantitative results are as Figure 2 and Figure 3As shown, a total of 4 rice disease resistance-related genes, namely OsPR1a, OsPR10, OsPAD4, and OsICS1, and 1 reference gene UBQ5-2 were detected in the experiment. Among them, OsPR1a is a downstream gene of induced resistance, and the others are disease resistance marker genes in the SA pathway. The leaf-induced resistance genes of Bacillus subtilis 168 and its ΔcomQ mutant were all significantly up-regulated compared with the CK, and OsPRIa, OSPRI0, and OsICS1 in the roots were significantly up-regulated compared with the CK, preliminarily indicating that it may have induced systemic disease resistance to rice through the SA pathway, and the disease resistance effect of ΔcomQ was more significant.

[0051] 2. Effects of Bacillus treatment on the ROS level in rice

[0052] Since reactive oxygen species in rice can act as local and systemic signaling molecules to induce plant disease resistance, in order to explore the relationship between reactive oxygen species in rice and induced resistance, ROS was measured in the roots of rice inoculated with each Bacillus.

[0053] Dilute the DCFH-DA kit with buffer to a concentration of 10 μM. After treating rice for 24 hours by the method in Example 2, sample the roots of rice. Immerse the samples of each treatment group in 10 μM DC-FDA, and vacuum infiltrate at 60 kPa for 5 min, and incubate in the dark at room temperature for 10 min. After incubation, wash the samples five times with double distilled water. Drop the positive control reagent on the positive control group and wait for 20 - 30 min to develop color. Place the samples on polylysine slides, blot off the excess moisture with absorbent paper, drop an appropriate amount of anti-fluorescence quencher in the center of the samples, and seal the slides with nail polish. After the slides are dried, observe the samples with a laser beam with an excitation wavelength of 488 nm under a confocal microscope and take pictures. Quantify the average fluorescence intensity of the rice roots taken with imagine J.

[0054] The ROS detection results of the root cap of rice roots are as Figure 4 shown. Compared with the CK, there was no difference between Bacillus subtilis 168 and the CK, but the average fluorescence intensity of the ΔcomQ mutant was significantly enhanced compared with its wild type.

[0055] The ROS detection results of the elongation zone of rice roots are as Figure 5 shown. Compared with the CK, the average fluorescence intensity of the elongation zone of the rice roots in the Bacillus subtilis 168 treatment group and the ΔcomQ mutant was significantly increased, and the enhancement effect of the ΔcomQ mutant was more significant. It shows that the ΔcomQ mutant can effectively increase the ROS level in rice, thereby enhancing the immune disease resistance ability of rice, that is, inducing plants to produce induced systemic resistance.

[0056] Example 3 Determination of the rice sheath blight resistance ability

[0057] To determine whether Bacillus can improve the rice's resistance to sheath blight, after treating the rice by the method of Example 2, in vitro pathogen inoculation and in vivo pathogen inoculation (the pathogen is Rhizoctonia solani Rs7) were carried out on the rice respectively.

[0058] (1) After 24 hours of treatment with the Bacillus bacterial solution, rice leaves of each treatment group were sampled, and the samples were placed flat in a humidity box. Mechanical damage was caused on the leaf surface with a needle. At the wound, a pathogen agar block with a diameter of 0.5 cm was uniformly placed. The humidity box was placed in a cool and lighted place, cultured for 5 - 6 days, the disease situation of the leaves was observed, the leaves were photographed, and the size of the rice disease spots was quantified using Image J.

[0059] (2) For the rice treated with the Bacillus bacterial solution for 24 hours, in vivo pathogen inoculation was carried out. Rice leaves with basically the same leaf size and growth condition were selected. Mechanical damage was caused on the rice leaves with a needle, and then a pathogen agar block with a diameter of 0.5 cm was uniformly placed at the wound. The agar block was fixed on the rice leaves with transparent tape and cultured under the conditions of 28 °C, 90% humidity, 12 - hour light, and 12 - hour darkness. After culturing for one week, the diseased leaves were observed, photographed, and the area of the rice disease spots was quantified using Image J.

[0060] The results are as Figure 6 and Figure 7 shown. Compared with the CK, the area of the disease spots of the rice treated with Bacillus subtilis 168 was significantly reduced. The rice leaves treated with the ΔcomQ mutant were not susceptible to the disease, and the Rs7 hyphae could only adhere to the surface of the rice leaves and could not invade from the mechanically damaged parts, indicating that the disease resistance of the rice treated with the ΔcomQ mutant was significantly enhanced.

[0061] Example 4 RNA-seq analysis of the expression differences of rice induced resistance-related genes and related pathways

[0062] To explore the changes in the disease resistance-related genes of rice after inoculation with Bacillus, the rice treated with the bacteria was sent for RNA-seq. The culture method of Bacillus was as in Example 2. One day after inoculating the rice leaves with Bacillus, a 7-day-old Rhizoctonia solani Rs7 plate was taken, and 10 mL of sterile water was injected into each plate. The fungal hyphae were gently scraped with a flat brush and mixed evenly with the sterile water. After the fungal mixture was collected, it was filtered on a medical gauze to make a hyphal suspension, and the suspension was immediately sprayed on the rice leaves of each treatment group. The rice treated with Rhizoctonia solani Rs7 was cultured under the conditions of 28 °C, 90% humidity, 12 - hour light, and 12 - hour darkness. The rice leaves after inoculation with Bacillus and after treatment with Rhizoctonia solani were taken for transcriptome sequencing.

[0063] The results are as Figure 8As shown, the transcriptome results indicate that after inoculating rice with the ΔcomQ mutant, pattern recognition receptors (PRRs) on the plant cell surface recognize the bacterial flagellin Flg22 to initiate an immune response. After activation, the PRRs cause the oligomers of the nonexpressor of pathogenesis-related genes 1 (NPR1) protein to depolymerize into monomers through SA synthesis and enter the nucleus. In the nucleus, the NPR1 protein interacts with the TGA transcription factor, binds to specific cis-acting elements in the promoter region of the PR1 gene, and activates the transcription of the PR1 gene, thereby promoting the expression of the PR1 protein. At the same time, in the MAPK pathway, MKK4 / 5 is activated, which in turn activates MPK3 / 6. Phosphorylated MPK3 / 6 acts on DNA to induce the expression of the PR1 gene and trigger a hypersensitive response. During this process, glutathione (GSH) continuously maintains the redox balance in rice cells, enabling them to maintain normal cell functions and structures when attacked by Rhizoctonia solani, thereby enhancing resistance to diseases. KEGG analysis shows that the MAPK pathway, salicylic acid pathway, ROS pathway, and glutathione pathway act synergistically when plants resist bacterial infections, ultimately inducing the expression of PR1 to resist pathogens.

[0064] The heatmap shows the expression levels of disease-resistant genes in the ΔcomQ mutant and its wild-type Bacillus subtilis 168 (Bs168) compared to CK after inoculating with pathogenic bacteria. The genes in each pathway of the ΔcomQ mutant were significantly upregulated, while there was no significant difference in Bs168 compared to CK, indicating that the ability of the ΔcomQ mutant to induce resistance in rice was significantly enhanced compared to the wild type, that is, it was proved that: the ComQ gene of Bacillus subtilis and the Bacillus subtilis 168 ΔcomQ mutant can improve the induced systemic resistance of plants.

Claims

1. Application of the comQ gene of the Bacillus subtilis ComQXPA quorum sensing system in enhancing the ability of Bacillus subtilis to induce systemic resistance in plants.

2. The application according to claim 1, characterized in that The said application is to inhibit the expression of the comQ gene in Bacillus subtilis or knockout the comQ gene in Bacillus subtilis.

3. Application of the Bacillus subtilis ΔcomQ mutant strain in enhancing plant induced systemic resistance.

4. The application according to claim 3, wherein The said Bacillus subtilis ΔcomQ mutant strain is the Bacillus subtilis 168ΔcomQ mutant strain.

5. The application according to claim 4, characterized in that The construction method of the said Bacillus subtilis 168ΔcomQ mutant strain is the homologous recombination method.

6. The application according to any one of claims 1 or 3, characterized in that The said enhancement of plant induced systemic resistance is to increase the expression level of plant disease resistance genes, the ROS level in plants and / or reduce the lesion area of plant leaves.

7. A method for enhancing plant induced systemic resistance to control plant diseases, characterized in that, Applying the bacterial suspension of the fermentation culture solution of the Bacillus subtilis ΔcomQ mutant strain to plants.

8. The method according to claim 7, wherein The said application method is spraying, root irrigation or root dipping.

9. The application according to any one of claims 1 or 3, or the method according to claim 7, characterized in that The said plant is rice.

10. According to the application as claimed in claim 1 or 3, or the method as claimed in claim 7, characterized in that The said plant disease is rice sheath blight.

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