A method for cloning an avirulence gene from a small chromosome of rice blast fungus

Abstract

The application discloses a method for cloning an avirulence gene from a small chromosome of Magnaporthe grisea, which comprises the following steps: (1) screening a strain carrying an avirulence gene corresponding to an anti-rice blast gene X; (2) performing ultraviolet mutagenesis on the screened strain; (3) spraying the strain after ultraviolet mutagenesis on a rice variety carrying the anti-rice blast gene X, and collecting the avirulence gene mutant strain; (4) performing mixed pool sequencing on the avirulence gene mutant strain obtained in the step (3); and (5) locating the avirulence gene through association analysis. The application does not depend on the fertility of the strain, sexual recombination and the genomic sequence polymorphism among different strains, and the target gene can be located through the association analysis by obtaining the avirulence gene mutant.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a method for cloning a non-toxic gene from a small chromosome of rice blast fungus. Background Technology

[0002] Rice is a major food crop in my country and Southeast Asian countries. Rice production is constantly threatened by rice blast. The annual yield reduction due to rice blast is enough to feed 60 million people. Currently, the main measures for rice blast control are cultivating resistant rice varieties and applying pesticides at appropriate times. Resistant varieties typically carry major blast resistance genes, mostly the NB-LRR gene. These genes interact with the avirulence gene (AVR) of the rice blast fungus to trigger a hypersensitive response, killing infection sites and hyphae, ultimately resulting in a resistant phenotype. Therefore, cloning the avirulence gene and detecting its distribution within the rice blast fungus population is crucial for the rational layout and rotation of resistant rice varieties.

[0003] Avirulence-free genes are essentially effectors of pathogens. They are secreted into the host cell or apoplast space, helping the pathogen successfully infect the host by suppressing the host's immune response or by seizing energy from the host. However, some effectors can be recognized by host blast resistance genes (usually encoding NB-LRR proteins), initiating an effector-triggered immunity (ETI) response. This response can cause cellular hypersensitivity, killing infection site cells and infection hyphae, inhibiting the colonization and proliferation of infection hyphae, and causing the plant to exhibit a disease-resistant phenotype. Most of the currently cloned avirulence-free genes encode cysteine-rich, small-molecule secretory proteins. They are preferentially distributed in rapidly evolving genomic regions rich in repetitive sequences such as transposon elements, including the protelomere regions of core chromosomes and independent small chromosomes.

[0004] Traditional gene cloning methods involve constructing sexually mated populations and locating the target gene based on trends in recombination events near it. However, small chromosomes are typically strain-specific, making it difficult to find homologous small chromosomes in two strains with different mating backgrounds. Furthermore, sexual recombination events are usually absent on small chromosomes within sexually mated populations. Therefore, it is impossible to clone non-toxic genes on small chromosomes using traditional methods. Summary of the Invention

[0005] The purpose of this invention is to provide a method for cloning avirulent genes from the small chromosome of rice blast fungus, which does not rely on strain fertility, sexual recombination, or genomic sequence polymorphism between different strains. By obtaining avirulent gene mutants, the target gene can be located through association analysis.

[0006] The technical solution adopted by this invention to solve its technical problem is:

[0007] A method for cloning a virus-free gene from a small chromosome of rice blast fungus includes the following steps:

[0008] (1) By taking advantage of the interaction between the non-toxic gene and the rice blast resistance gene, the strain carrying the non-toxic gene corresponding to the rice blast resistance gene X was screened by inoculating rice varieties carrying different rice blast resistance genes with rice blast fungus strains.

[0009] (2) The selected strains were subjected to ultraviolet mutagenesis;

[0010] (3) The strain after ultraviolet mutagenesis was sprayed onto rice varieties carrying the rice blast resistance gene X. If the strain after ultraviolet mutagenesis could infect rice varieties carrying the rice blast resistance gene X, it indicates that the non-toxic gene of the strain has mutated. Collect the non-toxic gene mutant strain.

[0011] (4) Perform pooled sequencing on the non-toxic gene mutant strains obtained in step (3);

[0012] (5) Locate non-toxic genes through association analysis.

[0013] The research object of this invention, the avirulence gene, is actually the gene locus in *Pseudomonas oryzae* that can be recognized by rice resistance genes. Only one gene locus is considered at a time. Through screening in step (1), strains containing the avirulence gene corresponding to the rice blast resistance gene are obtained. Screening in step (1) involves inoculating *Pseudomonas oryzae* strains onto rice varieties with different individual resistances; if infection occurs, it indicates the presence of the corresponding avirulence gene.

[0014] The purpose of UV mutagenesis is to induce mutations in the non-toxic gene. Although UV mutagenesis is random, the final result obtained through screening on the corresponding resistant rice in step (3) is a mutant of the target non-toxic gene, independent of other random mutation sites. That is, the result obtained through UV mutagenesis + targeted screening is a reproducible targeted result. Step (5) can locate the target gene based on the correlation between the mutation site and the trait.

[0015] by Pi4a For example, through vaccination Pi4a If the rice blast strain cannot infect Pi4a rice, the Guy11 strain can. This suggests that the rice blast strain carries a non-virulent gene. AvrPi4a The Guy11 strain does not carry the avirulence gene. AvrPi4a Next, the rice blast strain was subjected to ultraviolet mutagenesis, and the mutagenized strain was inoculated. Pi4a Rice. If the mutated strain can infect... Pi4a Rice, indicating its code. AvrPi4a A mutation occurred.

[0016] Preferably, in step (2), the UV mutagenesis of the selected strains is carried out as follows: the conidia of the selected strains are made into a bacterial suspension, the bacterial suspension is spread evenly in an uncovered petri dish, and placed under a 254nm UV lamp for 10-15s to carry out the mutagenesis. The UV lamp is positioned 10 cm above the bacterial suspension.

[0017] Preferably, the concentration of conidia in the bacterial suspension is 1×10⁻⁶. 6 per mL.

[0018] Preferably, in step (4), the strains used for pooled sequencing are subjected to REP-PCR molecular fingerprint analysis using Pot2 sequence primers to exclude contaminated strains.

[0019] As a preferred option, the information for the Pot2 sequence primers is: F: 5'-CGGAAGCCCTAAAGCTGTTT-3, R: 5'-CCCTCATTCGTCACACGTTC-3'.

[0020] Preferably, the rice blast resistance gene X is selected from... Pi4a One of the following genes: Pi1, Pi2, Pi3, Pi5, Pi7, Pi12, Pi19, Pi20, Pi21, Pigm, Pit, and Pish.

[0021] Preferably, in step (1), strains carrying a non-toxic gene corresponding to the rice blast resistance gene X are screened out, wherein the rice blast resistance gene X is... Pi4a The non-toxic gene is AvrPi4a This invention is also applicable to the cloning of non-toxic genes corresponding to other rice blast resistance genes, including non-toxic genes corresponding to Pi1, Pi2, Pi3, Pi5, Pi7, Pi12, Pi19, Pi20, Pi21, Pigm, Pit, and Pish genes.

[0022] The beneficial effects of this invention are: this invention does not rely on strain fertility, sexual recombination, or genomic sequence polymorphism between different strains. As long as a few non-toxic gene mutants are obtained, the target gene can be located through association analysis, which helps to quickly clone non-toxic genes from the small chromosome of rice blast fungus. Attached Figure Description

[0023] Figure 1 This is a flowchart illustrating the present invention.

[0024] Figure 2 The effect of different UV irradiation times on the germination rate of rice blast fungus conidia.

[0025] Figure 3These are the REP-PCR analysis results of 34 non-toxic gene mutants of rice blast fungus obtained by ultraviolet mutagenesis.

[0026] Figure 4 This study identifies the pathogenicity of non-toxic mutant strains of rice blast fungus obtained through ultraviolet mutagenesis on disease-resistant rice.

[0027] Figure 5 This is a flowchart of mixed-pool sequencing and data analysis of non-toxic mutant strains of rice blast fungus.

[0028] Figure 6 This is a statistical analysis of the distribution of deletion variations and deletion regions on the mini1 superchromosome of UV-mutated strains.

[0029] Figure 7 These are the results of functional verification of the candidate genes; Figure 7 The validation results for five candidate fragments are shown: 0-3 kb, 3-6 kb, 9-12 kb, 0-6 kb, and 6-12 kb. The results for 6-9 kb are not shown. 0-3k-1, 0-3k-2, and 0-3k-3 represent the results of three parallel settings in the 0-3 kb group, and the other numbers correspond to different groups as explained above. Detailed Implementation

[0030] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0031] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. Unless otherwise specified, the methods in the following embodiments are conventional methods in the field.

[0032] Test strains and plants:

[0033] The tested strain was wild-type rice blast fungus strain FJ81278 (carrying a non-virulence gene). AvrPi4a (Provided by the Fungal Laboratory of Fujian Agriculture and Forestry University) and Guy11 (which does not carry the avirulence gene) AvrPi4a (Provided by the Fungal Laboratory of Fujian Agriculture and Forestry University); the rice used in the test was the susceptible rice blast variety CO39 and... Pi4a Rice.

[0034] The process of this invention is as follows Figure 1 As shown.

[0035] Example 1: Obtaining non-toxic gene mutants through ultraviolet mutagenesis

[0036] (1) Inoculate the mycelium of rice blast fungus strain FJ81278 onto the surface of a 9 cm diameter rice bran medium (40 g / L rice bran, 20 g / L agar powder). Incubate in the dark at 28°C for five days. After the mycelium has fully colonized the medium, scrape off the surface mycelium and wash the conidia with sterile water into a 15 cm culture dish. To ensure that the conidia concentration is not less than 1 × 10⁻⁶ in subsequent spray inoculation experiments... 5 To balance mutagenesis efficiency and survival rate, the initial spore concentration was set at 1 × 10⁶ spores / mL. 6 per mL.

[0037] (2) The culture dish was transferred to a UV crosslinker (CX-2000 UV crosslinker, UVP, USA) for UV irradiation (254nm UV lamp). Six treatment gradients were set according to different UV irradiation times: 5 s, 10 s, 15 s, 20 s, 25 s and 30 s, with the unirradiated sample as the control.

[0038] (3) After UV irradiation, conidia were incubated at room temperature, fully moisturized, and in the dark for 24 h. Their germination rate was then calculated to assess the effect of UV irradiation. Figure 2 ).

[0039] The results showed that the germination rate of conidia decreased significantly with prolonged irradiation time. Figure 2 When the irradiation time was extended to 15 seconds, the germination rate dropped to about 10%, and after the irradiation time exceeded 20 seconds, all spores failed to germinate, exhibiting a completely inactive state. Based on a comprehensive analysis of the relationship between irradiation time and survival rate, 15 seconds of ultraviolet irradiation was determined to be the optimal mutagenesis condition.

[0040] Example 2: Screening using resistant rice AvrPi4a Non-toxic gene mutant strains

[0041] (1) Collection of conidia of rice blast fungus after ultraviolet irradiation: The conidia were resuspended in 0.002% (V / V) Tween 20 solution and the concentration was adjusted to 1×10⁻⁶. 6 per mL.

[0042] (2) Spray the conidial suspension onto plants at the two-leaf-one-heart stage. Pi4a Rice seedlings. Phenotypic analysis was conducted after 7 days of cultivation in a greenhouse (25℃, 70-80% humidity). This was because the rice blast fungus strain FJ81278 carries a non-virulent gene. AvrPi4a Cannot be contaminated Pi4a Rice. If the mutated spores can infect... Pi4a Rice, which indicates its AvrPi4a Gene mutations have occurred.

[0043] (3) Collected in Pi4a The lesions that appear on rice leaves are candidate non-toxic gene mutants.

[0044] (4) REP-PCR molecular fingerprinting was performed using Pot2 sequence primers (F: 5'-CGGAAGCCCTAAAGCTGTTT-3 (SEQ ID No. 1), R: 5'-CCCTCATTCGTCACACGTTC-3' (SEQ ID No. 2)) (Method reference: Peng Z et al. Genetic Variation of Magnaporthe oryzae Population in HunanProvince. J Fungi (Basel). 2023 Jul 23;9(7):776.), and screened strains with the same genetic background as the parent strain FJ81278. Figure 3 The results showed that the REP-PCR fingerprints of 27 candidate avirulent mutant strains (marked in red in the figure) were identical to those of FJ81278, indicating that they may be the true mutants. avrpi4a The mutant strains were selected for further research; the remaining candidate strains showed significant differences in REP-PCR bands compared to FJ81278, suggesting they were contaminated strains and were not used for further research. Based on these results, 27 candidate avirulent gene mutant strains with the same genetic background as FJ81278 were selected as materials for further research and were uniformly renumbered as V-Pi4a-1~27.

[0045] (5) 27 candidate non-toxic gene mutant strains were inoculated with rice bran to produce sporulations, which were then used for inoculation. Pi4a Rice, to determine whether the strain is effective against... Pi4a Rice has the ability to infect.

[0046] (6) Inoculation results were obtained for 26 strains, and the inoculation results showed that 24 of the candidate strains could infect. Pi4a Rice and the development of disease lesions AvrPi4a Candidate strains ( Figure 4 ).

[0047] Example 3: AvrPi4a Pooled sequencing of non-virulent mutant strains to locate candidate genes ( Figure 5 )

[0048] (1) For 24 plants Pi4a Rice strains exhibiting susceptibility to the disease were sequenced using second-generation Illumina sequencing.

[0049] (2) Perform quality control on the raw sequencing data. Use Trimmomatic software to remove adapter sequences and filter low-quality reads to obtain high-quality clean data.

[0050] (3) Subsequently, the clean data were aligned to the reference genome FJ81278 using BWA-MEM software, and the alignment results were sorted, filtered and repetitive sequences were removed using SAMtools.

[0051] (4) Use the GATK tool to perform single-sample variant detection (SNP and InDel call), and then perform variant merging and strict filtering through sample joint analysis.

[0052] (5) Finally, we performed manual visualization verification using IGV and used R language for data statistics and graphing.

[0053] Analysis revealed that most candidate strains exhibited varying degrees of deletion variation in the telomere region of the mini1 superchromosome (Figure 6). Specifically, strains V-Pi4a-2, V-Pi4a-3, V-Pi4a-12, V-Pi4a-18, V-Pi4a-20, and V-Pi4a-21 showed complete deletions of the mini1 genome sequence; strains V-Pi4a-7, V-Pi4a-26, and V-Pi4a-2 showed large deletions approximately 12 kb, 15 kb, and 13 kb post-telomere, respectively; while the deletions in V-Pi4a-17 and V-Pi4a-19 were relatively small, confined to a region approximately 10 kb post-telomere. Based on these combined results, the 0-10 kb post-telomere region of mini1 was identified as a potential key candidate region.

[0054] Example 4: Functional Verification Cloning AvrPi4a Non-toxic genes

[0055] To further locate toxicity-related genes, we divided this region into six candidate fragments: 0-3 kb, 3-6 kb, 6-9 kb, 9-12 kb, 0-6 kb, and 6-12 kb. These fragments were then analyzed in strain Guy11 (excluding...). AvrPi4a Heterologous expression was performed in [the following] (method reference: Zheng Y, et al. AVR1-CO39 is a predominant locus governing the broad avivulence of [the following]). Magnaporthe oryzae 2539 on cultivated rice ( Oryza sativaL.). MolPlant Microbe Interact. 2011 Jan;24(1):13-7.). The resulting transformed strain was then spray-inoculated onto Pi4a In resistant rice materials, the susceptible variety CO39 was used as a control. Results showed that transformants carrying 0-3 kb and 0-6 kb fragments... Pi4a In rice, the strains exhibited a disease-resistant response, while those carrying other fragments were all susceptible. Figure 7 The results indicated that the target gene was located within the 0-3 kb region. Further analysis of RNA-seq expression data revealed that only one gene with expression activity existed in the 0-3 kb region post-telomere of mini1. Ultimately, we successfully cloned the avirulence gene of rice blast fungus. AvrPi4a .

[0056] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. A method for cloning a virus-free gene from a small chromosome of rice blast fungus, characterized in that, Includes the following steps: (1) By taking advantage of the interaction between the non-toxic gene and the rice blast resistance gene, the strain carrying the non-toxic gene corresponding to the rice blast resistance gene X was screened by inoculating rice varieties carrying different rice blast resistance genes with rice blast fungus strains. (2) The selected strains were subjected to ultraviolet mutagenesis; (3) The bacterial suspension of the ultraviolet-mutated strain was sprayed onto rice varieties carrying the rice blast resistance gene X. If the ultraviolet-mutated strain could infect the rice varieties carrying the rice blast resistance gene X, it indicated that the non-toxic gene of the strain had mutated. The non-toxic gene mutant strain was collected. (4) Perform pooled sequencing on the non-toxic gene mutant strains obtained in step (3); (5) Locating avirulent genes through association analysis; In step (2), the UV mutagenesis of the selected strains is specifically carried out as follows: the conidia of the selected strains are made into a bacterial suspension, the bacterial suspension is spread on an uncovered petri dish, and placed under a 254nm UV lamp for 10-15s exposure to carry out mutagenesis; In step (1), strains carrying a virus-free gene corresponding to the rice blast resistance gene X are screened out, wherein the rice blast resistance gene X is... Pi4a The non-toxic gene is AvrPi4a .

2. The method according to claim 1, characterized in that, The concentration of conidia in the bacterial suspension was 1×10⁻⁶. 6 per mL.

3. The method according to claim 1, characterized in that, In step (4), the strains used for pool sequencing are subjected to REP-PCR molecular fingerprint analysis using Pot2 sequence primers to exclude contaminated strains.

4. The method according to claim 3, characterized in that, The primer information for Pot2 sequence is: F: 5'-CGGAAGCCCTAAAGCTGTTT-3, R: 5'-CCCTCATTCGTCACACGTTC-3'.

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