A method for efficiently detecting histone modification sites and abundance at the whole genome level in fungi

Abstract

This invention discloses a highly efficient method for detecting histone modification sites and abundance at the whole-genome level in fungi. The method provided by this invention for detecting the distribution and abundance of histone modifications throughout the genome of the tested fungi includes the following steps: collecting fungal hyphae, preparing protoplasts, and then extracting cell nuclei; performing CUT&Tag on the cell nuclei for the purpose of detecting histone modifications. This invention is the first to apply CUT&Tag technology to the detection of histone modification content and localization in fungi, providing an important tool for studying the relationship between fungal histone modifications and their growth and pathogenicity.

Description

Technical Field

[0001] This invention relates to a method for efficiently detecting histone modification sites and abundance at the whole genome level in fungi. Background Technology

[0002] Nucleosomes are the basic structural units of chromatin. A nucleosome consists of an octamer formed by two copies each of four histones (H2B, H2A, H3, and H4) and approximately 146 bp of DNA wrapped around it. Histones, as important components of the nucleosome, are one of the decisive factors determining the degree of chromatin packaging. The amino acids at the N-terminal tail of histones can undergo various modifications, including acetylation, methylation, and ubiquitination. These modifications affect the compactness and accessibility of chromatin by altering the charge of the nucleosome or recruiting epigenetic regulatory factors that influence chromatin structure, further affecting gene expression and thus participating in the regulation of physiological and developmental processes in eukaryotes. Histone modification is one of the most important epigenetic regulatory mechanisms for gene expression in eukaryotes. In recent years, with the discovery of more and more types of histone modifications and the elucidation of their specific regulatory mechanisms, this epigenetic regulatory mechanism has received increasing attention and has gradually become a hot topic in life science research.

[0003] Chromatin immunoprecipitation with sequencing (ChIP-Seq) is a traditional and important tool for studying histone modifications, widely used in epigenetic research in plants and animals. Its basic principle involves fixing histones to DNA with formaldehyde, followed by cell disruption, sonication to break down the DNA, precipitation of the protein-DNA complex using specific antibodies, decrosslinking to release the DNA, followed by DNA purification, library construction, and sequencing to obtain histone-specific modification sites. However, this technique has many limitations: it is time-consuming; requires large sample volumes; has a low signal-to-noise ratio; has poor reproducibility; and exhibits low enrichment in fungi.

[0004] To address the drawbacks of this technology, Henikoff published a new histone modification technique, CUT&Tag, in Nature Communications in 2019. This technique uses concanavalin A-coated magnetic beads to adsorb live cells and utilizes digitonin to increase cell membrane permeability. Target protein-specific antibodies (primary antibodies) enter the cell and bind to specifically modified histones. Secondary antibodies recognize the primary antibody and amplify the signal. The pA / G-Tn5 transposase binds to both the primary and secondary antibodies, and then... 2+Under activation, the DNA at both ends of the target protein is cleaved, and adapters are added simultaneously. Library construction can then be performed via PCR. Sequencing allows for the detection of target protein binding sites or histone modification levels at the whole-genome level. qPCR can also detect the relative binding amount of the protein at the target gene or the histone modification level. This method is short-cycle, has a high signal-to-noise ratio, good reproducibility, requires a small number of cells, and holds promise for single-cell analysis. This method allows for a more accurate reflection of intracellular modification levels by keeping cells in a live state.

[0005] Since its publication, this technology has been widely used in animal cells, and it has also been successfully applied in plants to detect histone modification levels and transcription factor binding sites via CUT & Tag. However, due to the presence of cell walls and secondary metabolites in fungi, the application of this technology in fungi is challenging, and there are currently no reports on its use in fungi. Histone modification plays an important role in the growth, development, and pathogenicity of fungi. Because the application of ChIP technology in fungi has significant drawbacks, a new method to replace it is urgently needed. Summary of the Invention

[0006] The purpose of this invention is to provide a method for efficiently detecting histone modification sites and abundance at the whole genome level in fungi.

[0007] This invention provides a method for detecting the distribution and abundance of histone modifications throughout the genome of a tested organism, characterized in that:

[0008] The organism in question is a fungus;

[0009] The method for detecting the distribution and abundance of histone modifications in the whole genome of the tested organism provided by the present invention is fungal CUT&Tag; compared with the traditional CUT&Tag, the difference of the fungal CUT&Tag is that the following step is added before adsorbing the cell nucleus: take fungal hyphae and prepare protoplasts.

[0010] The traditional CUT & Tag process includes the following steps:

[0011] (1) Extracting cell nuclei;

[0012] (2) Adsorption of the cell nucleus;

[0013] (3) Primary antibody incubation;

[0014] (4) Secondary antibody incubation;

[0015] (5) Incubation with pA / G-Tn5 transposase;

[0016] (6) Fragmentation: Activation of pA / G-Tn5 transposase;

[0017] (7) Library amplification: DNA libraries are obtained through library amplification;

[0018] (8) Perform high-throughput sequencing on the DNA library and obtain the distribution and abundance of histone modifications in the whole genome of the tested organism based on the sequencing results.

[0019] The fungal CUT&Tag includes the following steps in sequence:

[0020] (1) Take fungal hyphae and prepare protoplasts;

[0021] (2) Extract cell nuclei;

[0022] (3) Adsorption of the cell nucleus;

[0023] (4) Primary antibody incubation;

[0024] (5) Secondary antibody incubation;

[0025] (6) Incubation with pA / G-Tn5 transposase;

[0026] (7) Fragmentation: Activation of pA / G-Tn5 transposase;

[0027] (8) Library amplification: DNA libraries are obtained through library amplification;

[0028] (9) Perform high-throughput sequencing on the DNA library and obtain the distribution and abundance of histone modifications in the whole genome of the tested organism based on the sequencing results.

[0029] CUT&Tag: Cleavage Under Targets and Tagmentation.

[0030] Specifically, the adsorption of cell nuclei is achieved by using ConA beads to adsorb cell nuclei.

[0031] Specifically, the ConA beads are ConA Beads Pro.

[0032] The preparation of protoplasts includes the following steps: taking hyphae, and utilizing... Propionids are enzymatically digested and then collected.

[0033] Specifically, enzymatic hydrolysis is performed using an enzymatic hydrolysate; in the enzymatic hydrolysate, The Pro content is 2g / 100ml.

[0034] Specifically, the enzymatic hydrolysis conditions are: 33°C, 60 rpm shaking reaction for 3 hours.

[0035] Specifically, the enzymatic hydrolysis conditions are: 33°C, 60 rpm shaking reaction for 30 min.

[0036] Specifically, the preparation of protoplasts includes the following steps: take hyphae, place them in an enzymatic hydrolysate, and react with shaking at 33°C and 60 rpm for 30 min to 3 h (specifically, 30 min or 3 h); then, filter with a double-layer magic filter cloth, collect the filtrate, centrifuge the filtrate at 3800 rpm for 10 min, discard the supernatant, and wash with 0.7 M NaCl aqueous solution.

[0037] Enzymatic hydrolysate: Take 0.25g VinoTaste Pro, dissolve it in 12.5ml of 0.7M NaCl aqueous solution, rotate to dissolve for 20min, then filter it through a 0.22μm pore size filter membrane and collect the filtrate.

[0038] Specifically, the preparation of protoplasts includes the following steps: take hyphae, place them in an enzymatic hydrolysate, and react with shaking at 33°C and 60 rpm for 30 min to 3 h (specifically, 30 min or 3 h); then, filter with a double-layer magic filter cloth, collect the filtrate, centrifuge the filtrate at 3800 rpm for 10 min, discard the supernatant, and wash with SCS solution.

[0039] Enzymatic hydrolysate: Take 0.25g Pro was dissolved in 12.5 ml of SCS solution and vortexed for 20 min. The solution was then filtered through a 0.22 μm pore size membrane, and the filtrate was collected.

[0040] Specifically, the primary antibody incubation is performed using a primary antibody working solution.

[0041] Specifically, the primary antibody is incubated for 16 hours.

[0042] Specifically, the secondary antibody incubation is performed using a secondary antibody working solution.

[0043] Specifically, the incubation time for the secondary antibody is 60 minutes.

[0044] The specific incubation time for the pA / G-Tn5 transposase can be 60 minutes.

[0045] The pA / G-Tn5 transposase consists of a pA / G-Tn5 fusion protein and a linker. The pA / G-Tn5 fusion protein is a fusion protein of Tn5 transposase and Protein A / G. The linker binds to the Tn5 transposase. Specifically, the pA / G-Tn5 transposase is pA / G-TnpPro. Specifically, the pA / G-Tn5 transposase is incubated with pA / G-Tn5 transposase working solution. pA / G-Tnp Pro working solution: Mix 2 μl of pA / G-Tnp Pro with 98 μl of Dig-300 Buffer.

[0046] Activation of pA / G-Tn5 transposase was achieved by adding TTBL working solution.

[0047] TTBL working solution: Mix 40 μl Dig-300 Buffer and 10 μl 5×TTBL.

[0048] Specifically, the CUT&Tag uses the following kit: Hyperactive Universal CUT&Tag AssayKit for Illumina Pro.

[0049] The CUT&Tag adsorbs the nuclei of 250,000 cells.

[0050] Specifically, the method for extracting cell nuclei includes the following steps:

[0051] Take 1×10 6 One protoplast was added to 100 μL of NE buffer and lysed on ice for 10 min. Then, it was centrifuged at 2500 rpm for 5 min, the supernatant was discarded, and the precipitate was resuspended in 100 μL of Wash Buffer to obtain the resuspension.

[0052] Mix 25 μl of resuspension with 75 μl of wash buffer to obtain 100 μl of cell nucleus suspension, which will be used for subsequent cell nucleus adsorption steps.

[0053] Specifically, the histone modification is H3K9me3, H3K27me3, H3K4me3, H3K36me3, or H3K27ac.

[0054] Specifically, the primary antibody is H3K9me3 antibody, H3K27me3 antibody, H3K4me3 antibody, H3K36me3 antibody, or H3K27ac antibody.

[0055] Specifically, the secondary antibody is Goat anti Rabbit IgG.

[0056] Specifically, the fungus is a filamentous fungus.

[0057] Specifically, the fungus is Verticillium dahliae, Neurospora crassa, or Ustilago maydis.

[0058] Fungi possess cell walls and abundant secondary metabolites, making it difficult for digitonin, used in traditional CUT&Tag technology, to directly permeate the fungal cell membrane. This invention successfully applies CUT&Tag-seq technology to fungi by adding a pretreatment step (i.e., preparing protoplasts before extracting the cell nucleus), enabling the detection of various histone modification levels and providing an important tool for histone modification research in fungi.

[0059] Compared to traditional ChIP technology, this invention has the following advantages:

[0060] ① The number of cells required is small, only 250,000 cells;

[0061] ② It is quick and easy to operate, and can be completed in just two days;

[0062] ③ High signal-to-noise ratio and low background noise;

[0063] ④ Good repeatability.

[0064] This invention is the first to apply CUT&Tag technology to detect the content and location of histone modifications in fungi, providing an important means for studying the relationship between fungal histone modifications and their growth and pathogenicity. Attached Figure Description

[0065] Figure 1 This is the result of the length distribution of the DNA library in Example 3.

[0066] Figure 2 The sequence results are from the DNA library in Example 3.

[0067] Figure 3 This is the result of the length distribution of the DNA library in Example 5.

[0068] Figure 4 The sequencing results are for the DNA library in Example 5.

[0069] Figure 5 The sequencing results are for the DNA library in Example 6.

[0070] Figure 6 The sequencing results are for the DNA library in Example 7.

[0071] Figure 7 The sequencing results are for the DNA library in Example 8. Detailed Implementation

[0072] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0073] Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Unless otherwise specified, the quantitative experiments in the following examples are all performed in triplicate, and the results are averaged. Miracloth: pore size 22-25 μm, Merck Millipore, catalog number 475855-1R. *Verticillium dahliae* V592, PGKO-HPT vector, and PGKO-NAT-AGO1 vector were kindly provided by the research group of Professor Guo Huishan at the Institute of Microbiology, Chinese Academy of Sciences. Recombinant strain Nc△dim5 was obtained from the research group of Professor He Qun at China Agricultural University and was donated by Professor Liu Xiao at the Institute of Microbiology, Chinese Academy of Sciences. *Neurospora crassa* 87-3 was donated by the research group of Professor Liu Xiao at the Chinese Academy of Sciences. Haploid strains JG35 and JG36 of *Ustilago maydis* were isolated physiological races from the Guangxi Academy of Agricultural Sciences and were donated by Professor Lu Shan of Guangxi University.

[0074] Hyperactive Universal CUT&Tag Assay Kit for Illumina Pro: Novizan Pharmaceuticals, catalog number TD904-01. The kit provides the following components: NE buffer, 10×Wash Buffer, 10×Binding Buffer, ConA Beads Pro, Antibody Buffer (-), pA / G-Tnp Pro (2μM), 10×Dig-300 Buffer, 5×TTBL, DNA Spike-in (5ng / μl), DNA Extract Beads Pro, 2×B&W Buffer, 2×CAM, 10% SDS, 5% Digitonin. 50× Protease Inhibitor: Dissolve one tablet of protease inhibitor (Roche, 04693132001) in 1 ml ddH2O. Binding Buffer: Mix 30 μl of 10×Binding Buffer with 270 μl of ddH2O. Wash Buffer: Mix 150 μl of 10× Wash Buffer, 30 μl of 50× protease inhibitor, and 1320 μl of ddH2O. Antibody Buffer: Take 50 μl of Antibody Buffer (-), add 0.5 μl of 5% Digitonin, mix well, and pre-cool on ice. Dig-wash Buffer: Mix 792 μl of Wash Buffer with 8 μl of 5% Digitonin. Dig-300 Buffer: Take 100 μl of 10× Dig-300 Buffer, add 2 μl of 5% Digitonin and 20 μl of 50× protease inhibitor, add 878 μl of ddH2O, and mix well. 1× B&W Buffer: Mix 500 μl of 2× B&W Buffer with 500 μl of ddH2O.

[0075] Example 1: Establishment of the Method

[0076] I. Preparation of protoplasts

[0077] 1. Take a 100mL Erlenmeyer flask, add the mycelium of the test fungus and 10mL of enzyme hydrolysate, and shake at 33℃ and 60rpm for reaction.

[0078] Enzymatic hydrolysate: Take 0.25g Pro was dissolved in 12.5 ml of 0.7 M NaCl aqueous solution and rotated for 20 min. Then, it was filtered through a 0.22 μm pore size filter membrane, and the filtrate was collected. Pro (product form is solid powder): Novozyme Corporation.

[0079] 2. After completing step 1, filter the solution using a double-layered magic filter cloth, collect the filtrate, centrifuge the filtrate at 3800 rpm for 10 min, discard the supernatant, wash the precipitate with 0.7M NaCl aqueous solution, and then resuspend the precipitate in 10 ml of 0.7M NaCl aqueous solution. This is the protoplast solution (count the protoplasts using a hemocytometer).

[0080] II. Obtaining the cell nucleus

[0081] 1. Take 1×10 6 One protoplast was added to 100 μL of NE buffer and lysed on ice for 10 min. Then, it was centrifuged at 2500 rpm for 5 min, the supernatant was discarded, and the precipitate was the cell nucleus.

[0082] 2. After completing step 1, add 100 μl of Wash Buffer to resuspend the precipitate, which is the resuspension solution.

[0083] 3. Mix 25 μl of the resuspended solution obtained in step 2 with 75 μl of wash buffer to obtain 100 μl of cell nucleus suspension.

[0084] III. Preparation of DNA Libraries Using CUT & Tag Technology

[0085] 1. ConA beads treatment

[0086] ① Take an 8-tube bundle, add 100 μl of Binding Buffer to each tube, then add 10 μl of ConABeads Pro to each tube, mix well, then place the 8-tube bundle on a magnetic rack, and discard the supernatant after the solution becomes clear.

[0087] ②After completing step ①, remove the 8-tube set from the magnetic rack, add 100μl of Binding Buffer to each tube, mix well, then place the 8-tube set back on the magnetic rack. After the liquid has clarified, discard the supernatant.

[0088] ③ After completing step ②, add 10 μl of Binding Buffer to each tube to resuspend ConA Beads Pro.

[0089] 2. The cell nucleus binds to ConA beads during incubation.

[0090] Take the 8-tube set from step 1, add 100 μl of cell nuclear suspension to each tube, invert to mix, and then incubate at room temperature for 10 min (invert to mix 2-3 times during this period). After a brief centrifugation, place the 8-tube set on a magnetic rack and discard the supernatant after the liquid has cleared.

[0091] 3. Primary antibody incubation

[0092] Take the 8-tube set from step 2, add 50 μl of pre-cooled primary antibody working solution to each tube, invert to mix, centrifuge briefly, and then let stand at 4°C for 16 h.

[0093] 4. Secondary antibody incubation

[0094] ① Take the 8-tube set from step 3, centrifuge briefly, then place it on a magnetic rack. After the liquid becomes clear, discard the supernatant.

[0095] ② Take the 8-tube set from step ①, add 50 μl of secondary antibody working solution to each tube, invert to mix, and incubate at room temperature for 60 min.

[0096] ③ Take the 8-tube set from step ②, centrifuge briefly, then place the 8-tube set on a magnetic rack. Wait for the liquid to clarify, then discard the supernatant.

[0097] ④ Take the 8-tube strip from step ③, add 200 μl of Dig-wash Buffer to each tube, invert to mix, centrifuge briefly, place the 8-tube strip on a magnetic rack, wait for the liquid to clarify, and discard the supernatant.

[0098] ⑤ Take the 8-tube strip from step ④, add 200 μl of Dig-wash Buffer to each tube, invert to mix, centrifuge briefly, place the 8-tube strip on a magnetic rack, wait for the liquid to clarify, and discard the supernatant.

[0099] ⑥ Take the 8-tube strip from step ⑤, add 200 μl of Dig-wash Buffer to each tube, invert to mix, centrifuge briefly, place the 8-tube strip on a magnetic rack, wait for the liquid to clarify, and discard the supernatant.

[0100] 5. pA / G-Tnp incubation

[0101] ① Take the 8-tube set from step 4, add 100 μl of pA / G-Tnp Pro working solution to each tube, invert to mix, and incubate at room temperature for 60 min.

[0102] pA / G-Tnp Pro working solution: Mix 2 μl of pA / G-Tnp Pro with 98 μl of Dig-300 Buffer.

[0103] ② Take the 8-tube set from step ①, centrifuge briefly, place the 8-tube set on a magnetic rack, wait for the liquid to clarify, and discard the supernatant.

[0104] ③ Take the 8-tube strip from step ②, add 200 μl of Dig-300 Buffer to each tube, invert to mix, centrifuge briefly, place the 8-tube strip on a magnetic rack, wait for the liquid to clarify, and discard the supernatant.

[0105] ④ Take the 8-tube strip from step ③, add 200 μl of Dig-300 Buffer to each tube, invert to mix, centrifuge briefly, place the 8-tube strip on a magnetic rack, wait for the liquid to clarify, and discard the supernatant.

[0106] ⑤ Take the 8-tube strip from step ④, add 200 μl of Dig-300 Buffer to each tube, invert to mix, centrifuge briefly, place the 8-tube strip on a magnetic rack, wait for the liquid to clarify, and discard the supernatant.

[0107] 6. Fragmentation

[0108] ① Take the 8-tube set from step 5, add 50 μl of TTBL working solution to each tube, invert to mix, and incubate at 37°C for 60 min.

[0109] TTBL working solution: Mix 40 μl Dig-300 Buffer and 10 μl 5×TTBL.

[0110] ② Take the 8 tubes from step ①, centrifuge briefly, add 2 μl of 10% SDS and DNA Spike-in with a DNA content of 2.5 pg to each tube, and incubate at 55℃ for 10 min (invert and mix 2-3 times during this period).

[0111] ③ After completing step ②, centrifuge briefly, place the 8-tube set on a magnetic rack, let stand for about 2-3 minutes, and carefully transfer the supernatant to a new 8-tube set.

[0112] 7. DNA Extract Beads Pro treatment

[0113] ① Take 25 μl of DNA Extract Beads Pro into a 1.5 ml centrifuge tube, add 200 μl of 1×B&W Buffer, mix well, place on a magnetic rack, and discard the supernatant after the solution becomes clear.

[0114] ②After completing step ①, remove the 1.5ml centrifuge tube from the magnetic rack, add 200μl of 1×B&W Buffer, mix well, place it on the magnetic rack, and discard the supernatant after the solution becomes clear.

[0115] ③ After completing step ②, add 50 μl of 2×B&W Buffer to a 1.5 ml centrifuge tube to resuspend DNA ExtractBeads Pro, which is the DNA ExtractBeads Pro suspension.

[0116] 8. DNA extraction

[0117] ① Take the 8-tube containing the supernatant obtained in step 6, add 50 μl of DNA ExtractBeads Pro suspension obtained in step 7 to each tube, invert to mix, and incubate at room temperature for 20 min (inverting 2-3 times during this period).

[0118] ②After completing step ①, centrifuge briefly, place the 8-tube set on a magnetic rack, and discard the supernatant after the solution has clarified.

[0119] ③ After completing step ②, keep the 8-tube strip on the magnetic rack at all times, add 200 μl of 1×B&W Buffer to each tube, incubate at room temperature for 30 seconds, and discard the supernatant.

[0120] ④ After completing step ③, keep the 8-tube strip on the magnetic rack at all times, add 200 μl of 1×B&W Buffer to each tube, incubate at room temperature for 30 seconds, and discard the supernatant.

[0121] ⑤ After completing step ④, open the lid and let it air dry at room temperature for 2-5 minutes until there is no liquid residue in the tube and the surface of the magnetic beads is no longer reflective.

[0122] ⑥ After completing step ⑤, remove the 8-tube bundle from the magnetic rack and add 15 μl of ddH2O to each tube to resuspend the DNAExtractBeads Pro.

[0123] 9. Library expansion

[0124] Prepare the amplification system according to Table 1, and then perform amplification according to Table 2.

[0125] Table 1

[0126] volume The resuspension prepared in step 8 15μl 2×CAM 25μl N5XX 5μl N7XX 5μl Total volume 50μl

[0127] Table 2

[0128]

[0129] TruePrep Index Kit V2 for Illumina: Novizan Corporation, catalog number TD202. The TruePrep Index Kit V2 for Illumina provides 8 N5XX (N501-N508) and 12 N7XX (N701-N712) types. Table 1 shows that any one of the N5XX and one of the N7XX types can be selected.

[0130] 10. Purify and recover DNA

[0131] ① After completing step 9, add 100 μl of VAHTSDNA Clean Beads (Vazyme#N411) to each reaction tube, mix well, incubate at room temperature for 5 min, centrifuge briefly, then place the reaction tube on a magnetic rack, and carefully remove the supernatant after the solution has clarified.

[0132] ②After completing step ①, keep the reaction tubes on the magnetic rack, add 200μl of 80% ethanol aqueous solution to each tube, incubate at room temperature for 30 seconds, and carefully remove the supernatant.

[0133] ③ After completing step ②, keep the reaction tubes on the magnetic rack, add 200 μl of 80% ethanol aqueous solution to each tube, incubate at room temperature for 30 seconds, carefully remove the supernatant, and open the cap to dry for 3-5 minutes.

[0134] ④ After completing step ③, remove the reaction tubes from the magnetic rack, add 22 μl of ddH2O to each tube for elution, mix well, incubate at room temperature for 5 min, centrifuge briefly, then place the reaction tubes on the magnetic rack. After the solution becomes clear, carefully aspirate 20 μl of supernatant into a new EP tube, which is the DNA library solution.

[0135] IV. Sequencing

[0136] 1. Take the DNA library solution obtained in step 3, and use the Qubit dsDNA HS Assay Kit to perform quantification according to the instructions. Use Qseq 400 to detect the peak distribution.

[0137] 2. Take the DNA library solution obtained in step four and perform sequencing.

[0138] The library was sequenced using the paired-end 150bp sequencing mode on the Illumina Novaseq 6000 sequencing platform.

[0139] Example 2: Construction of recombinant bacteria Vd△kmt1

[0140] KMT1: Histone H3K9me3 methyltransferase KMT1.

[0141] The KMT1 gene in the genomic DNA of Verticillium dahliae V592 is shown in sequence 1 of the sequence listing.

[0142] Verticillium dahliae V592 and pGKO-HPT vector are described in the following literature: Wang S, Xing H, Hua C, Guo HS, Zhang J. An Improved Single-Step Cloning Strategy Simplifies the Agrobacterium tumefaciens-Mediated Transformation (ATMT)-Based Gene-Disruption Method for Verticillium dahliae. Phytopathology. 2016 Jun; 106(6):645-52. doi:10.1094 / PHYTO-10-15-0280-R. Epub 2016 Mar 30. PMID:26780432.

[0143] 1. Using the pGKO-HPT vector as the starting vector, an upstream homologous arm was inserted into the upstream PacI restriction site of the HPT gene (as shown in positions 1-1037 of Sequence 2 in the sequence listing), and a downstream homologous arm was inserted into the downstream PacI restriction site of the HPT gene (as shown in positions 3535-4537 of Sequence 2 in the sequence listing), resulting in a recombinant plasmid. Sequencing verification confirmed that the recombinant plasmid contained the DNA molecule shown in Sequence 2 of the sequence listing. In Sequence 2 of the sequence listing, positions 1-1037 form the upstream homologous arm, positions 1445-2470 encode the hygromycin resistance protein (HPT), and positions 3535-4537 form the downstream homologous arm.

[0144] 2. The recombinant plasmid obtained in step 1 was introduced into Agrobacterium EHA105 to obtain recombinant Agrobacterium.

[0145] 3. The recombinant Agrobacterium obtained in step 2 was co-cultured with Verticillium dahliae V592 spores, and then recombinant Verticillium dahliae was obtained by screening with hygromycin and named recombinant strain Vd△kmt1. Compared with the genomic DNA of Verticillium dahliae V592, the only difference in the genomic DNA of recombinant strain Vd△kmt1 is that the KMT1 gene has been replaced with the DNA molecule shown in positions 1038-3534 of sequence 2 in the sequence listing.

[0146] Example 3: Detection of the distribution and abundance of H3K9me3 in Verticillium dahliae V592 and recombinant strain Vd△kmt1

[0147] The tested fungi were Verticillium dahliae V592 (denoted as WT) or recombinant strain Vd△kmt1.

[0148] I. Preparation of Mycelium

[0149] 1. Activation of strains: Inoculate the test fungi stored at -80℃ onto PDA medium plates and incubate at 26℃ for 3-4 days.

[0150] 2. Spore culture: After completing step 1, pick up a piece of mycelium from the plate and inoculate it into Czapek's culture medium. Incubate at 26°C and 220 rpm for 24 hours with shaking to obtain spore culture medium.

[0151] Czapek's medium (pH 7.2): Dissolve 30g sucrose, 3g NaNO3, 0.5g MgSO4·7H2O, 0.5g KCl, 100mg FeSO4·7H2O and 1g K2HPO4 in ddH2O and bring the volume to 1L.

[0152] 3. Mycelial culture: Inoculate 5 mL of the spore culture solution prepared in step 2 into 500 mL of liquid YEPD medium and culture at 26℃ and 220 rpm for 18 h with shaking.

[0153] Liquid YEPD medium: Take 3g yeast extract, 10g peptone, and 20g glucose, dissolve them in ddH2O and bring the volume to 1L.

[0154] 4. After completing step 3, filter and collect the mycelium using the magic filter cloth. First wash it 3 times with ddH2O, then wash it 3 times with 0.7M NaCl aqueous solution.

[0155] II. Detection of the distribution and abundance of H3K9me3 at the fungal genome level

[0156] Take the mycelium prepared in step one and test it according to the method in Example 1.

[0157] In step 1 of Example 1, the oscillation reaction time is 3 hours.

[0158] The primary antibody working solution was the H3K9me3 antibody working solution. The H3K9me3 antibody working solution was prepared by diluting the H3K9me3 antibody to a 50-fold volume using Antibody Buffer. The H3K9me3 antibody (Histone H3K9me3 antibody) (rabbit-derived, liquid form): Active Motif, catalog number 39161.

[0159] Secondary antibody working solution: Take the secondary antibody and dilute it 100 times with Dig-wash Buffer. Secondary antibody (Goat antiRabbit IgG): Novizan Pharmaceuticals, catalog number AB207-01-AA.

[0160] The results of the DNA library length distribution are shown in [the table]. Figure 1 The DNA library solutions all met sequencing requirements.

[0161] Sequencing results of the DNA library are shown below. Figure 2 In the method provided by this invention, H3K9me3 is well enriched. After knocking out the KMT1 gene, the level of H3K9me3 decreases.

[0162] Example 4: Construction of recombinant bacteria Vd△ezh2

[0163] EZH2: H3K27me3 methyltransferase EZH2.

[0164] The EZH2 gene in the genomic DNA of Verticillium dahliae V592 is shown in sequence 3 of the sequence listing.

[0165] The PGKO-NAT-AGO1 vector is described in the following literature: Wen, HG., Zhao, JH., Zhang, BS. et al. Microbe-induced gene silencing boosts crop protection against soil-borne fungal pathogens. Nat. Plants 9, 1409–1418 (2023). https: / / doi.org / 10.1038 / s41477-023-01507-9.

[0166] 1. Using the PGKO-NAT-AGO1 vector as the starting vector, an upstream homologous arm was inserted into the PacI restriction site upstream of the NAT gene (as shown in positions 1-1020 of Sequence 4 in the sequence listing), and a downstream homologous arm was inserted into the PacI restriction site downstream of the NAT gene (as shown in positions 2364-3288 of Sequence 4 in the sequence listing), resulting in a recombinant plasmid. Sequencing verification confirmed that the recombinant plasmid contained the DNA molecule shown in Sequence 4 of the sequence listing. In Sequence 4, positions 1-1020 form the upstream homologous arm, positions 1390-1959 encode N-acetyltransferase (NAT), and positions 2364-3288 form the downstream homologous arm.

[0167] 2. The recombinant plasmid obtained in step 1 was introduced into Agrobacterium EHA105 to obtain recombinant Agrobacterium.

[0168] 3. The recombinant Agrobacterium obtained in step 2 was co-cultured with Verticillium dahliae V592 spores, and then recombinant Verticillium dahliae was obtained through NAT screening, named recombinant strain Vd△ezh2. Compared with the genomic DNA of Verticillium dahliae V592, the only difference in the genomic DNA of recombinant strain Vd△ezh2 is that the EZH2 gene has been replaced with the DNA molecule shown in positions 1021-2363 of sequence 4 in the sequence listing.

[0169] Example 5: Detection of the distribution and abundance of H3K27me3 in Verticillium dahliae V592 and recombinant strain Vd△ezh2. The tested fungi were Verticillium dahliae V592 (represented by WT) or recombinant strain Vd△ezh2.

[0170] I. Preparation of Mycelium

[0171] Same as step one in Example 3.

[0172] II. Detection of the distribution and abundance of H3K27me3 at the fungal genome level

[0173] Take the mycelium prepared in step one and test it according to the method in Example 1.

[0174] In step 1 of Example 1, the oscillation reaction time is 3 hours.

[0175] The primary antibody working solution was the H3K27me3 antibody working solution. The H3K27me3 antibody working solution was prepared by diluting the H3K27me3 antibody to a 50-fold volume using Antibody Buffer. The H3K27me3 antibody (Histone H3K27me3 antibody) (rabbit-derived, liquid form, 1 μg / μl): Active Motif, catalog number 39155.

[0176] Secondary antibody working solution: Take the secondary antibody and dilute it 100 times with Dig-wash Buffer. Secondary antibody (Goat antiRabbit IgG): Novizan Pharmaceuticals, catalog number AB207-01-AA.

[0177] The results of the DNA library length distribution are shown in [the table]. Figure 3 The DNA library solutions all met sequencing requirements.

[0178] Sequencing results of the DNA library are shown below. Figure 4 In the method provided by this invention, H3K27me3 is well enriched. After knocking out the EZH2 gene, the level of H3K27me3 decreases.

[0179] Example 6: Detection of the distribution and abundance of H3K4me3, H3K36me3 or H3K27ac in Verticillium dahliae. The tested fungus was Verticillium dahliae V592.

[0180] I. Preparation of Mycelium

[0181] Same as step one in Example 3.

[0182] II. Detection of the distribution and abundance of fungal genome-level H3K4me3, H3K36me3, or H3K27ac

[0183] Take the mycelium prepared in step one and test it according to the method in Example 1.

[0184] In step 1 of Example 1, the oscillation reaction time is 3 hours.

[0185] The primary antibody working solutions were H3K4me3 antibody working solution, H3K36me3 antibody working solution, H3K27ac antibody working solution, or IgG antibody working solution (as controls).

[0186] H3K4me3 antibody working solution: Dilute the H3K4me3 antibody to 50-fold volume with Antibody Buffer to obtain the H3K4me3 antibody working solution. H3K4me3 antibody (Histone H3K4me3 antibody) (rabbit-derived, liquid product): Active Motif, catalog number 39060.

[0187] H3K36me3 antibody working solution: Dilute the H3K36me3 antibody to 50-fold volume with Antibody Buffer to obtain the H3K36me3 antibody working solution. H3K36me3 antibody (Histone H3K36me3 antibody) (rabbit-derived, liquid product, specification 1μg / μl): Active Motif, catalog number 61902.

[0188] H3K27ac antibody working solution: Dilute the H3K27ac antibody to 50-fold volume with Antibody Buffer to obtain the H3K27ac antibody working solution. H3K27ac antibody (Histone H3K27ac antibody) (rabbit source, liquid form, specification 1μg / μl: Active Motif, catalog number 39034).

[0189] IgG antibody working solution: Dilute Rabbit IgG control Polyclonal antibody to 50-fold volume with Antibody Buffer to obtain IgG antibody working solution. Rabbit IgG control Polyclonal antibody (product form: liquid): Proteintech, catalog number 30000-0-AP.

[0190] Secondary antibody working solution: Take the secondary antibody and dilute it 100 times with Dig-wash Buffer. Secondary antibody (Goat antiRabbit IgG): Novizan Pharmaceuticals, catalog number AB207-01-AA.

[0191] Sequencing results of the DNA library are shown below. Figure 5 Using the method provided by this invention, H3K4me3 / H3K36me3 / H3K27ac all achieved good enrichment effects.

[0192] Example 7: Detection of Neurospora crassa

[0193] Neurospora crassa is an important eukaryotic model organism, playing a crucial role in genetics and molecular biology research. Neurospora crassa 87-3 (the wild-type strain in the literature) and the recombinant strain Nc△dim5 (dim-5KO in the literature) are described in the following literature: Zhao Y, Shen Y, Yang S, Wang J, Hu Q, Wang Y, He Q. Ubiquitin ligase components Cullin4 and DDB1 are essential for DNA methylation in Neurosporacrassa. J Biol Chem. 2010 Feb 12; 285(7):4355-65. doi:10.1074 / jbc.M109.034710. Epub2009Nov 30.PMID:19948733;PMCID:PMC2836040. Compared to Neurospora crassa 87-3, the recombinant strain Nc△dim5 has the dim5 gene knocked out.

[0194] The tested fungi were Neurospora crassa 87-3 (denoted as WT) or recombinant Nc△dim5.

[0195] The hyphae of the tested fungus were taken and tested according to the method in Example 1.

[0196] In step 1 of Example 1, the oscillation reaction time is 30 min.

[0197] The primary antibody working solution was the H3K9me3 antibody working solution. The H3K9me3 antibody working solution was prepared by diluting the H3K9me3 antibody to a 50-fold volume using Antibody Buffer. The H3K9me3 antibody (Histone H3K9me3 antibody) (rabbit-derived, liquid form): Active Motif, catalog number 39161.

[0198] Secondary antibody working solution: Take the secondary antibody and dilute it 100 times with Dig-wash Buffer. Secondary antibody (Goat antiRabbit IgG): Novizan Pharmaceuticals, catalog number AB207-01-AA.

[0199] Sequencing results of the DNA library are shown below. Figure 6 H3K9me3 was well enriched in *Neurospora crassa* 87-3, while the level of H3K9me3 decreased in the recombinant strain NcΔdim5. The method provided by this invention is applicable to the detection of histone modifications in *Neurospora crassa*.

[0200] Example 8: Detection of Sugarcane Ustilago maydis

[0201] Sugarcane smut is the pathogen of sugarcane smut, one of the most significant diseases affecting sugarcane production worldwide. Histone modification plays a crucial role in the pathogenic process of this fungus, thus a simple and reliable method for detecting histone modification is urgently needed. The haploids JG35 and JG36 of sugarcane smut are described in the following literature: Lan Xianruan, Wang Yukun, Wu Haoming, Lu Shan, Qiu Jinfeng, Li Ru, Chen Baoshan. Early visual observation of sugarcane smut infection [J]. Acta Microbiologica Sinica, 2021, 61(3):596-606.

[0202] The tested fungi were haploid JG35 or haploid JG36 of *Ustilago maydis*.

[0203] I. Preparation of protoplasts

[0204] 1. Pick the test fungi and incubate them on YEPS liquid medium at 28°C with shaking at 200 rpm for 1 day, then transfer them to YEPS liquid medium (initial OD). 600nm ≈0.05), cultured at 28℃ and 200rpm with shaking until OD600. 00nm ≈0.7, then centrifuge at 1000g for 5 min, collect the bacterial cells, wash once with SCS solution, centrifuge at 1000g for 5 min at room temperature, and collect the bacterial cells.

[0205] YEPS liquid medium (pH 6.3): 10g yeast extract, 20g peptone, 20g sucrose, add deionized water to a final volume of 1L.

[0206] 2. Take a 100mL Erlenmeyer flask, add the bacterial cells obtained in step 1 and 10mL of enzyme hydrolysate, and shake at 33℃ and 60rpm for 30min.

[0207] Enzymatic hydrolysate: Take 0.25g Pro was dissolved in 12.5 ml of SCS solution and vortexed for 20 min. The solution was then filtered through a 0.22 μm pore size membrane, and the filtrate was collected. Pro (product form is solid powder): Novozyme Corporation.

[0208] SCS solution (pH 5.8): contains 20 mmol / L trisodium citrate and 1 mol / L sorbitol, with the remainder being water.

[0209] 3. After completing step 2, centrifuge at 1000g for 10 minutes, discard the supernatant, wash the precipitate with SCS solution, and then resuspend the precipitate with 5mL of SCS solution to obtain the protoplast solution (count the protoplasts using a hemocytometer).

[0210] The subsequent steps are the same as steps two, three and four of Example 1.

[0211] The primary antibody working solution was either H3K9me3 antibody working solution, H3K27me3 antibody working solution, or IgG antibody working solution (as a control).

[0212] H3K9me3 antibody working solution: Dilute the H3K9me3 antibody to 50-fold volume with Antibody Buffer to obtain the H3K9me3 antibody working solution. H3K9me3 antibody (Histone H3K9me3 antibody) (rabbit-derived, liquid product): Active Motif, catalog number 39161.

[0213] H3K27me3 antibody working solution: Dilute the H3K27me3 antibody to 50-fold volume with Antibody Buffer to obtain the H3K27me3 antibody working solution. H3K27me3 antibody (Histone H3K27me3 antibody) (rabbit-derived, liquid product, specification 1μg / μl): Active Motif, catalog number 39155.

[0214] IgG antibody working solution: Dilute Rabbit IgG control Polyclonal antibody to 50-fold volume with Antibody Buffer to obtain IgG antibody working solution. Rabbit IgG control Polyclonal antibody (product form: liquid): Proteintech, catalog number 30000-0-AP.

[0215] Secondary antibody working solution: Take the secondary antibody and dilute it 100 times with Dig-wash Buffer. Secondary antibody (Goat antiRabbit IgG): Novizan Pharmaceuticals, catalog number AB207-01-AA.

[0216] Sequencing results of the DNA library are shown below. Figure 7 The results showed that H3K9me3 and H3K27me3 were both well enriched, indicating that the method provided by this invention can be used to detect the histone modification level of *Ustilago maydis*, providing a new method for subsequent studies on the impact of epigenetic modifications on the pathogenicity of *Ustilago maydis*.

[0217] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for detecting the distribution and abundance of histone modifications in the whole genome of a tested fungus, comprising the following steps in sequence: (1) Take fungal hyphae and enzymatically hydrolyze them using VinoTaste® Pro to obtain protoplasts; (2) Extracting cell nuclei; (3) Adsorption of the cell nucleus; (4) Primary antibody incubation; (5) Secondary antibody incubation; (6) Incubation with pA / G-Tn5 transposase; (7) Fragmentation: Activation of pA / G-Tn5 transposase; (8) Library amplification: DNA libraries are obtained through library amplification; (9) Perform high-throughput sequencing on the DNA library and obtain the distribution and abundance of histone modifications in the whole genome of the tested organism based on the sequencing results; The fungus is Verticillium dahliae, Neurospora crassa, or Ustilago canaliculata.

2. The method as described in claim 1, characterized in that: The method adsorbs the nuclei of 250,000 cells.

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