A method for constructing a library of large fragment deletion mutants of magnaporthe grisea

Abstract

The application discloses a method for constructing a large fragment deletion mutant library of Magnaporthe oryzae, and belongs to the field of agricultural biotechnology.The method comprises the step of adding DEB to the protoplast of the Magnaporthe oryzae for culture and inducing the production of conidia.The application utilizes the chemical mutagen DEB to obtain the Magnaporthe oryzae mutant at a high frequency on a whole genome scale, and provides a new technical path for the functional genomics research of the Magnaporthe oryzae.The method provided by the application is helpful to systematically analyze the pathogenic mechanism, the infection process and the environmental adaptation regulation network of the Magnaporthe oryzae, and provides support for mining new disease-resistant targets and disease-resistant mechanisms.The method has the advantages of high efficiency, low cost and short cycle, and has wide application potential in the genetic research of plant pathogenic fungi.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and in particular to a method for constructing a library of large fragment deletion mutants of rice blast fungus. Background Technology

[0002] Currently, methods for obtaining mutant libraries from plant fungi mainly include physical mutagenesis, chemical mutagenesis, and biological mutagenesis. Physical mutagenesis is represented by ultraviolet (UV) radiation and ionizing radiation (γ-rays, X-rays), which primarily lead to characteristic imprints such as C→T base transition mutations. Chemical mutagenesis mainly uses ethyl methanesulfonate (EMS), which primarily leads to G:C→A:T base transition mutations. Biological mutagenesis mainly includes Agrobacterium-mediated random integration of T-DNA (ATMT) technology and CRISPR-Cas9 editing technology. ATMT technology is widely used in constructing filamentous fungal mutant libraries, while CRISPR-Cas9-mediated precise editing is only suitable for studying the editing of known or conserved gene sequences.

[0003] However, in practical applications, due to differences in actual environment and genetic background, as well as the rapid reproduction cycle of fungi, the genome sequences of the same filamentous fungus can vary significantly, making it extremely difficult to locate the target gene corresponding to a specific biological phenotype using traditional mutagenesis methods. The chemical mutagen DEB has been widely used in the construction of human cells and rice mutants. Compared with traditional mutagenesis methods and EMS chemical mutagenesis, DEB mutagenesis is significantly more efficient than traditional mutagenesis methods, including EMS chemical mutagenesis. Furthermore, DEB treatment not only induces single-base mutations in the sequence, but more than half of the mutations are long fragment deletions of approximately 1 kb, a characteristic that enables rapid location and cloning of the target gene. Summary of the Invention

[0004] The purpose of this invention is to provide a method for constructing a library of large fragment deletion mutants of *Magnaporum oryzae*, in order to solve the problems existing in the prior art. This invention targets the protoplast cells of *Magnaporum oryzae* and uses the chemical mutagen DEB to obtain *Magnaporum oryzae* mutants at a high frequency on a whole-genome scale, providing a new technical path for functional genomics research on *Magnaporum oryzae*.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for efficiently constructing a library of large fragment deletion mutants of rice blast fungus, including the step of adding DEB to the protoplasts of rice blast fungus for culturing and inducing the production of conidia.

[0006] Optionally, the following steps are included: DEB was added to the protoplasts of the rice blast fungus, and the mixture was allowed to stand in the dark. Then, liquid TB3 medium was added to revive the protoplasts and culture them to obtain the protoplast revival solution. Pour unsolidified oat medium into a petri dish and allow it to solidify. At the same time, mix the protoplast resuscitation solution with another unsolidified oat medium and pour it into the petri dish containing solidified oat medium prepared above. First, place it in the dark for incubation, and then continue to incubate under light to induce the production of conidia. Collect conidia to obtain the large fragment deletion mutant library of *Oryza sativa*.

[0007] Optionally, the volume fraction of the DEB is 0.1%, and the volume ratio of the protoplast to the DEB is 1 mL:1 μL.

[0008] Optionally, the conditions for the light-protected standing are 28°C and 24-36 h. The liquid TB3 culture medium consisted of 3 g yeast extract, 3 g acid-hydrolyzed complexin, 200 g sucrose, and distilled water to a final volume of 1 L. The conditions for the resuscitation culture were 28℃ and 80 rpm for 10-12 h.

[0009] Optionally, the oat culture medium consists of: 30 g of ground oat flakes, 15 g of agar, and distilled water to a final volume of 1 L; The protoplast resuscitation solution was brought to a final volume of 50 mL using the other unconsolidated oat culture medium.

[0010] Optionally, the culture time under dark conditions is 2 days, and the culture time under continuous light is 5-7 days.

[0011] The present invention also provides the application of the large fragment deletion mutant library of rice blast fungus constructed according to the method in rice disease resistance breeding.

[0012] The present invention also provides the application of the large fragment deletion mutant library of rice blast fungus constructed according to the method in screening and locating non-virulent genes of rice blast fungus.

[0013] The present invention also provides the application of the large fragment deletion mutant library of rice blast fungus constructed according to the method in the study of the interaction mechanism between rice blast fungus and rice.

[0014] The present invention discloses the following technical effects: 1) The mutation type is mainly large fragment deletion, with a high degree of functional mutation enrichment. More than half of the mutants generated by DEB mutagenesis are large deletion mutations of about 1 kb (Wu JL, Wu C, Lei C, Baraoidan M, Bordeos A, Madamba MR, Ramos-Pamplona M, Mauleon R, Portugal A, Ulat VJ, Bruskiewich R, Wang G, Leach J, Khush G, Leung H. Chemical- and irradiation-induced mutants of indica rice IR64 for forward and reverse genetics. Plant Mol Biol. 2005 Sep;59(1):85-97. doi: 10.1007 / s11103-004-5112-0. PMID: 16217604.), while traditional mutagenesis methods mainly use point mutations (Shirasawa K, HirakawaH, Nunome T, Tabata S, Isobe S. Genome-wide survey of artificial mutations induced by ethyl methanesulfonate and Gamma rays in tomato. Plant Biotechnol J. 2016 Jan;14(1):51-60. doi: 10.1111 / pbi.12348. Epub 2015 Feb 16. PMID:25689669; PMCID: PMC5023996.). Point mutations are affected by codon degeneracy, and mutations at key domains can only affect gene function. However, long deletions can directly lead to gene loss of function, which is beneficial for quickly obtaining phenotypically significant functional mutants.

[0015] 2) High efficiency and low noise in target gene localization Large deletion mutations can be quickly identified by whole-genome alignment, significantly narrowing the scope of localization and avoiding the screening interference caused by a large number of "non-functional mutations" in traditional point mutations, thus greatly improving the screening efficiency and accuracy of target genes.

[0016] 3) It facilitates the discovery of novel functional genes in unannotated regions. Traditional point mutations are difficult to identify effectively in unannotated regions of the genome, while the long deletion mutations generated by this invention can directly label unannotated regions. Combined with bioinformatics analysis, they can efficiently discover potential new functional genes and regulatory elements.

[0017] 4) The mutant library can be expanded indefinitely, making it suitable for high-throughput functional genomics research. The mutant strains obtained by this method can be stably inherited and continuously propagated, making them suitable for constructing a large-scale mutant resource library for long-term use, and providing a stable, high-throughput technical platform for functional genomics research on rice blast fungus.

[0018] 5) Low cost, simple operation, and strong versatility This method requires no complex instruments or equipment and can be completed under conventional experimental conditions. It has low experimental costs and low technical barriers, and is suitable for genetic research on a variety of plant pathogenic fungi. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram illustrating the application of the large fragment deletion mutant library of rice blast fungus. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] The application of disease-resistant varieties is the most economical and effective strategy for controlling rice blast in rice production. Its theoretical basis comes from the "gene-to-gene" theory, that is, the disease-resistant genes in rice ( R The gene can specifically recognize the non-virulence gene in the rice blast fungus. Avr The gene product triggers a rapid and strong immune response, preventing pathogen infection. Currently, there are over 100 rice varieties... R Genes have been located, and more than 40 of them have been successfully cloned and are undergoing functional studies. However, in stark contrast, only about 10 genes in the rice blast fungus remain unidentified. Avr The severe asymmetry in gene identification and cloning has greatly limited the systematic analysis of the interaction mechanism between rice and rice blast, and has also restricted, to some extent, [further research / exploration]. R Precise application of genes in disease-resistant breeding.

[0027] Based on the chemical mutagenesis strategy proposed in this invention, it is possible to efficiently obtain materials capable of breaking through specific limits. R Gene-mediated resistance mutant strains of rice blast fungus. Compared to traditional field isolation or random insertion mutagenesis methods, this strategy tends to generate large deletion mutations, thus significantly increasing the yield of mutant strains. Avr The probability of loss-of-function mutants. Furthermore, the genetic background differences among field isolates are enormous; even without artificial mutagenesis, their genomes exhibit a large amount of natural variation, often introducing numerous non-functionally relevant candidate regions into differential analysis, severely interfering with the results. Avr Precise gene localization. In contrast, this method cannot infect specific genes. R Using genetically modified materials and model strains with complete reference genomes (such as 70-15 and Guy11) as genetic backgrounds, whole-genome comparative analysis of strains before and after mutagenesis was performed. The backgrounds were highly consistent, allowing all differential fragments to be directly used as functional candidate regions, fundamentally reducing screening noise and improving localization accuracy.

[0028] Furthermore, by combining specific R-gene rice materials with spray inoculation screening, and purifying and verifying the pathogenicity of only a very small number of lesion strains formed on resistant materials, rice blast fungus mutant strains with key pathogenic functions can be selectively enriched. The genome of these mutants inevitably contains elements related to... Avr Deletion or mutation of functionally related genes or regulatory regions. By performing genome-wide differential alignment with wild-type strains, candidate strains can be rapidly identified within a relatively small genome. Avr Genes, possessing advantages such as small localization range, high accuracy, and fast verification efficiency, are crucial for understanding rice blast fungi. Avr The systematic mining of genes and the study of rice disease resistance mechanisms provide a high-throughput, low-noise, and highly scalable technical paradigm.

[0029] The chemical mutagen 1,2:3,4-diepoxybutane (DEB) used in this embodiment is a commercial product purchased from ThermoFisher (catalog number L03919).

[0030] Oat culture medium composition: 30 g of ground oat flakes, 15 g of agar, and distilled water to a final volume of 1 L.

[0031] Liquid TB3 medium: 3 g yeast extract, 3 g acid-hydrolyzed complexin, 200 g sucrose, and distilled water to a final volume of 1 L.

[0032] Genetically modified rice NPB- Piz-t Provided by Researcher Kang Houxiang from the Chinese Academy of Agricultural Plant Protection.

[0033] Example 1 A method for efficiently and rapidly constructing a large fragment deletion mutant library of *Oryza sativa* includes the following steps: 1) After conventional mycelial culture of the model strain Guy11 of Magnaporthe oryzae, protoplasts of Magnaporthe oryzae were extracted and prepared (for the preparation of protoplasts of Magnaporthe oryzae, refer to Chen, Y., Le, X., Sun, Y., Li, M., Zhang, H., Tan, X., Zhang, D., Liu, Y. and Zhang, Z. (2017), MoYcp4 is required for growth, conidiogenesis and pathogenicity in Magnaporthe oryzae. Molecular Plant Pathology, 18: 1001-1011.).

[0034] 2) DEB mutagenesis method for rice blast fungus protoplasts: i. Preparation: Prepare a 0.1% (v / v) DEB / 0.7 M NaCl stock solution and a 55℃ non-solidified oat culture medium; ii. Mutagenesis treatment: Add 1 μL of 0.1% DEB / 0.7 M NaCl to 1 mL of protoplasts (the higher the concentration, the greater the number of mutant libraries obtained), and let stand at 28°C in the dark for 24 h; iii. Add 4 mL of liquid TB3 medium to the mutagenesis tube and incubate at 28°C and 80 rpm in a constant temperature shaker for 10 h. iv. Pour 50 mL of unsolidified oat culture medium into a 120 mm petri dish and allow it to solidify (at the bottom of the petri dish). Meanwhile, the protoplast resuscitation solution was brought to a final volume of 50 mL using another portion of unsolidified oat medium, mixed thoroughly, and then poured into the culture dish containing solidified oat medium prepared above (located on the upper layer of the culture dish). v. Induce conidia production by placing the culture medium containing protoplasts in the dark for 2 days, followed by continuous light culture for 5 days; vi. Collect conidia of rice blast fungus to obtain a library of large fragment deletion mutants of rice blast fungus.

[0035] Example 2 A method for efficiently and rapidly constructing a large fragment deletion mutant library of *Oryza sativa* includes the following steps: 1) After routine mycelial culture of the rice blast fungus strain 70-15, rice blast fungus protoplasts were routinely extracted and prepared (the preparation of rice blast fungus protoplasts refers to Chen, Y., Le, X., Sun, Y., Li, M., Zhang, H., Tan, X., Zhang, D., Liu, Y. and Zhang, Z. (2017), MoYcp4 is required for growth, conidiogenesis and pathogenicity in Magnaporthe oryzae. Molecular Plant Pathology, 18: 1001-1011.).

[0036] 2) DEB mutagenesis method for rice blast fungus protoplasts: i. Preparation: Prepare a 0.1% (v / v) DEB / 0.7 M NaCl stock solution and a 55℃ non-solidified oat culture medium; ii. Mutagenesis treatment: Add 1 μL of 0.1% DEB / 0.7 M NaCl to 1 mL of protoplasts (the higher the concentration, the greater the number of mutant libraries obtained), and incubate at 28°C in the dark for 36 h; iii. Add 4 mL of liquid TB3 medium to the mutagenesis tube and incubate at 28°C and 80 rpm in a constant temperature shaker for 12 h. iv. Pour 50 mL of unsolidified oat culture medium into a 120 mm petri dish and allow it to solidify (at the bottom of the petri dish). Meanwhile, the protoplast resuscitation solution was brought to a final volume of 50 mL using another portion of unsolidified oat medium, mixed thoroughly, and then poured into the culture dish containing solidified oat medium prepared above (located on the upper layer of the culture dish). v. Induce conidia production by placing the culture medium containing protoplasts in the dark for 2 days, followed by continuous light culture for 7 days; vi. Collect conidia of rice blast fungus to obtain a library of large fragment deletion mutants of rice blast fungus.

[0037] Example 3 A method for efficiently and rapidly constructing a large fragment deletion mutant library of *Oryza sativa* includes the following steps: 1) After routine mycelial culture of the rice blast fungus type strain P131, rice blast fungus protoplasts were routinely extracted and prepared (the preparation of rice blast fungus protoplasts refers to Chen, Y., Le, X., Sun, Y., Li, M., Zhang, H., Tan, X., Zhang, D., Liu, Y. and Zhang, Z. (2017), MoYcp4 is required for growth, conidiogenesis and pathogenicity in Magnaporthe oryzae. Molecular Plant Pathology, 18: 1001-1011.).

[0038] 2) DEB mutagenesis method for rice blast fungus protoplasts: i. Preparation: Prepare a 0.1% (v / v) DEB / 0.7 M NaCl stock solution and a 55℃ non-solidified oat culture medium; ii. Mutagenesis treatment: Add 1 μL of 0.1% DEB / 0.7 M NaCl to 1 mL of protoplasts (the higher the concentration, the greater the number of mutant libraries obtained), and let stand at 28°C in the dark for 30 h; iii. Add 4 mL of liquid TB3 medium to the mutagenesis tube and incubate at 28°C and 80 rpm in a constant temperature shaker for 11 h. iv. Pour 50 mL of unsolidified oat culture medium into a 120 mm petri dish and allow it to solidify (at the bottom of the petri dish). Meanwhile, the protoplast resuscitation solution was brought to a final volume of 50 mL using another portion of unsolidified oat medium, mixed thoroughly, and then poured into the culture dish containing solidified oat medium prepared above (located on the upper layer of the culture dish). v. Induce conidia production by placing the culture medium containing protoplasts in the dark for 2 days, followed by continuous light culture for 6 days; vi. Collect conidia of rice blast fungus to obtain a library of large fragment deletion mutants of rice blast fungus.

[0039] Application examples It is known that strain Guy11 carries a non-virulence gene. AvrPiz-t Normal spray inoculation cannot infect transgenic rice NPB- Piz-t .like Figure 1 As shown, according to the method of Example 1, a large amount of Guy11 mutant library conidial suspension was obtained by DEB chemical mutagenesis treatment, and NPB- was spray-inoculated in batches. Piz-t Genetically modified rice allows mutant strains from the library to infect NPB- Piz- t Genetically modified rice can be isolated from its leaf margins in small numbers of lesions. These lesions only... AvrPiz-t Only strains with mutations or deletions can form this pattern. The process involves purifying and culturing the lesion strain, sequencing its entire genome, and performing a genome-wide sequence difference analysis with Guy11. Because the mutant genome contains a deletion of approximately 1 kb, the target band cannot be amplified using PCR. Therefore, it is sufficient to amplify the missing long fragment in the mutant from the Guy11 genome sequence, perform PCR amplification and sequencing analysis, and preliminarily locate the candidate sequence.

[0040] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for efficiently constructing a library of large fragment deletion mutants of *Oryza sativa*, characterized in that, The method includes the step of adding DEB to the protoplasts of the rice blast fungus and culturing them to induce the production of conidia.

2. The method as described in claim 1, characterized in that, Includes the following steps: DEB was added to the protoplasts of the rice blast fungus, and the mixture was allowed to stand in the dark. Then, liquid TB3 medium was added to revive the protoplasts and culture them to obtain the protoplast revival solution. Pour unsolidified oat medium into a petri dish and allow it to solidify. At the same time, mix the protoplast resuscitation solution with another unsolidified oat medium and pour it into the petri dish containing solidified oat medium prepared above. First, place it in the dark for incubation, and then continue to incubate under light to induce the production of conidia. Collect conidia to obtain the large fragment deletion mutant library of *Oryza sativa*.

3. The method as described in claim 2, characterized in that, The volume fraction of DEB is 0.1%, and the volume ratio of protoplast to DEB is 1 mL:1 μL.

4. The method as described in claim 2, characterized in that, The conditions for light-protected standing are 28℃ and 24-36h. The composition of the liquid TB3 culture medium is as follows: 3 g yeast extract, 3 g acid-hydrolyzed complexin, 200 g sucrose, and distilled water to a final volume of 1 L. The conditions for the resuscitation culture were 28℃ and 80 rpm for 10-12 h.

5. The method as described in claim 2, characterized in that, The oat culture medium consists of: 30 g of ground oat flakes, 15 g of agar, and distilled water to a final volume of 1 L. The protoplast resuscitation solution was brought to a final volume of 50 mL using the other unconsolidated oat culture medium.

6. The method as described in claim 2, characterized in that, The culture time under dark conditions is 2 days, and the culture time under continuous light is 5-7 days.

7. The application of the large fragment deletion mutant library of rice blast fungus constructed by the method described in any one of claims 1-6 in rice disease resistance breeding.

8. The application of the large fragment deletion mutant library of *Strombus oryzae* constructed by the method described in any one of claims 1-6 in screening and locating avirulent genes of *Strombus oryzae*.

9. The application of the large fragment deletion mutant library of *Magnaporum oryzae* constructed by the method described in any one of claims 1-6 in the study of the interaction mechanism between *Magnaporum oryzae* and rice.

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