This work was supported by National Natural Science Foundation of China (Grant no. 31871638), the Special Scientific Research Project of Beijing Agriculture University (YQ201603), the Scientific Project of Beijing Educational Committee (KM201610020005), the High-level scientific research cultivation project of BUA (GJB2021005).

1. Preparation of protoplasts from M. oryzae
<ol>
	<li>Prepare the oatmeal-tomato agar (OTA).
	<ol>
		<li>Weigh 30-50 g of oatmeal and boil it in 800 mL of water (ddH2O) for 20 min. Filter through two layers of gauze and take the filtrate.</li>
		<li>Pick ripe tomatoes and peel them. Squeeze the juice, and filter through two layers of gauze to collect 150 mL of the filtered juice.</li>
		<li>Mix all the tomato juice and the prepared oat filtrate thoroughly and add ddH2O up to 1000 mL.</li>
		<li>Add 250 mL of the OTA and 2.5 g of agar powder to a 500 mL conical flask, and autoclave for 20 min. Store at 25 &#176;C.</li>
	</ol>
	</li>
	<li>Pour 25 mL of the autoclaved OTA into a glass 5 cm x 5 cm Petri dish. Prepare 10 of these Petri dishes total. After the OTA has solidified on the Petri dishes, store the dishes upside down at 25 &#176;C.</li>
	<li>Use a sterilized toothpick to dig out a small piece of mycelium from M. oryzae (the wild-type strain P131, knockout strains &#916;mobre2, &#916;mospp1, and &#916;moswd2) and place them on the prepared OTA dishes. Culture them for 4-6 days at 28 &#176;C under light conditions. 
	NOTE: Turn the Petri dish upside down to prevent pollution.</li>
	<li>Add 1000 &#181;L of liquid Complete Medium (CM) (0.6% yeast extract, 0.3% enzymatic casein hydrolysate, 0.3% acidic casein hydrolysate, 1% glucose) to the OTA dishes using a 1000 &#181;L pipette. 
	NOTE: The hyphae grew on the OTA dishes for 4-6 days.</li>
	<li>Scrape the mycelia of the wild-type strain and knockout strain with an inoculation loop.</li>
	<li>Collect the mycelia debris and transfer them to 250 mL of liquid Complete Medium (CM).</li>
	<li>Grow the fungal debris in a triangular flask at 28 &#176;C for 36 h with shaking at 150 rpm.</li>
	<li>Use a funnel to filter and collect the fungal hyphae.</li>
	<li>Wash the fungal hyphae with 500 mL of 0.7 M NaCl solution.</li>
	<li>Collect the fungal mycelium and weigh it. 
	NOTE: The mycelium does not need to be dried before weighing.</li>
	<li>Add ~1 mL of lysis enzyme permeation solution per 1 g of the fungal mycelium.
	<ol>
		<li>Prepare the 20 mg/mL lysis enzyme permeation solution by dissolving the lysis enzyme from Trichoderma harzianum in 0.7 M NaCl.</li>
	</ol>
	</li>
	<li>Place the hyphae for lysing at 28 &#176;C for 3-4 h with shaking at 150 rpm.</li>
	<li>Wash the lysed hyphae with 50 mL of 0.7 M NaCl solution.</li>
	<li>Collect the protoplasts and centrifuge for 15 min at 2,000 x g and 4 &#176;C.</li>
	<li>After centrifugation, discard the supernatant carefully. Resuspend the protoplasts in 20 mL of 0.7 M NaCI buffer at 4 &#176;C.</li>
</ol>
2. In vivo crosslinking and sonication
<ol>
	<li>Add 55 &#181;L of 37% formaldehyde (add formaldehyde drop by drop until the final concentration is 1%) to 2 mL of NaCI buffer containing protoplast for crosslinking.</li>
	<li>Incubate the protoplasts at25 &#176;Cfor 10 min.</li>
	<li>Add 20 &#181;L of 10x glycine to each tube to quench the unreacted formaldehyde.</li>
	<li>Swirl to mix and incubate at 25 &#176;C for 5 min.</li>
	<li>Centrifuge for 15 min at 2,000 x g and 4 &#176;C.</li>
	<li>After centrifugation, discard the supernatant carefully. Resuspend the pellet in 1 mL of 0.7 M NaCI solution.</li>
	<li>Centrifuge for 10 min at 2,000 x g and 4 &#176;C.</li>
	<li>After centrifugation, discard the supernatant carefully. Resuspend the pellet in 750 &#181;L of RIPA buffer(50 mM Tris-HCl pH 8, 150 mM NaCl, 2 mM EDTA pH 8, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors).</li>
	<li>Add 37.5 &#181;L of 20x protease inhibitor.</li>
	<li>Transfer the protoplasts from the previous step to a 1.5 mL centrifuge tube.</li>
	<li>Perform sonication (25% W, output 3 s, stop 5 s, 4 &#176;C) immediately with a sonicator for about 10 min. The purpose of this step is to sonicate the cross-linked lysate for a period of time to determine the best conditions. 
	NOTE: The lysate can be frozen at -80 &#176;C at this step.</li>
	<li>If optimal conditions for sonication have already been determined, proceed to the next step.</li>
	<li>If desired, remove 5 &#181;L of protoplasts for agarose gel analysis (unsheared DNA).</li>
	<li>Shear the chromatin by sonication with an ultrasonic homogenizer for 8 min (25% W, output 3 s, stop 5 s, 4 &#176;C).</li>
	<li>After the sample is ultrasonically broken, take out a part of the sample as &#34;input&#34;.. The input does not perform the ChIP experiment and contains all DNA and protein released after the sample is sonicated.</li>
	<li>After sonication, run a 1% agarose gel electrophoresis to analyze the length of the DNA fragments. 
	NOTE: The agarose gel electrophoresis results show that the length of the DNA fragment is 200-500 bp (Figure 4).</li>
	<li>Place the sonicated tube on ice to prevent protein degradation.</li>
	<li>Centrifuge for 10 min at 10,000 x g and 4 &#176;C.</li>
	<li>After centrifugation, transfer the centrifuged supernatant to a new 1.5 mL centrifuge tube and store it at -80 &#176;C for later use. The chromatin solution obtained in this step can be used for subsequent IP.</li>
	<li>Before performing the IP experiment, dilute each chromatin sample to a ratio of 1:10 with 1x RIPA buffer (e.g., add 10 &#181;L of chromatin sample to 1 &#181;L of 1&#215;RIPA buffer).</li>
</ol>
3. IP of crosslinked protein/DNA
<ol>
	<li>Pipette 50 &#181;L of superparamagnetic protein beads into a 2 mL centrifuge tube. Place the tubes on a magnetic stand. Let the magnetic beads precipitate. Remove the supernatant.</li>
	<li>Add 1 mL of 1x RIPA buffer (pre-cooled on ice) to the tube and wash superparamagnetic protein beads thrice. After the wash, place the tubes on a magnetic stand and remove the supernatant. Add 100 &#181;L of 1x RIPA buffer to each tube.</li>
	<li>Add 300 &#181;L of chormatin sample (2 x 107 cells were used), 100 &#181;L of superparamagnetic protein beads and 4 &#181;L of H3K4me3 antibody to the tube.</li>
	<li>Use samples with Mouse IgG as the negative control. 
	NOTE: Mouse IgG used in this protocol contains 0.01 M phosphate buffer and 0.15 M NaCl, and will remain frozen below 20 &#176;C (see Table of Materials).</li>
	<li>After mixing well, place the samples on a rotary shaker and incubate overnight at 4 &#176;C, 30 x g. 
	&#8203;NOTE: It may be possible to reduce the incubation time of the immunoprecipitation (IP). The incubation time depends on different factors (e.g., the antibody, gene target, cell type, etc.) and will need to be empirically tested.</li>
</ol>
4. Collecting and rinsing the IP products
<ol>
	<li>Pellet the superparamagnetic protein beads by placing them on a magnetic stand. Aspirate and discard the supernatant.</li>
	<li>Wash the superparamagnetic protein bead-antibody/chromatin complex by resuspending the beads in 1 mL of 1x RIPA buffer.</li>
	<li>Rinse the tube on a rotary shaker for 5 min and remove the supernatant at 30 x g.</li>
	<li>Add 1 mL of low salt immune complex wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, and 150 mM NaCl).</li>
	<li>Rinse the tube on a rotary shaker for 5 min and remove the supernatant.</li>
	<li>Add 1 mL of high salt immune complex wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, and 500 mM NaCl) to the centrifuge tube.</li>
	<li>Place the tube on a rotary shaker for 5 min and remove the supernatant.</li>
	<li>Rinse with 1 mL of LiCl (0.25 M LiCl, 1% NP-40, 1% deoxycholic acid, 1 mM EDTA, and 10 mM Tris-HCl pH 8.1) in the centrifuge tube.</li>
	<li>Place the tube on a rotary shaker for 5 min and remove the supernatant.</li>
	<li>Rinse the test tube again with 1 mL of 0.25 M LiCI buffer, remove the supernatant with a pipette, and then discard the supernatant.</li>
	<li>Add 1 mL of TE buffer (10 mM Tris-HCl pH 8.1, 1 mM EDTA).</li>
	<li>Place the tube on a rotary shaker for 5 min at 30 x g.</li>
	<li>Wash the tube again with 1 mL TE buffer and remove the supernatant with a pipette.</li>
	<li>Collect the beads.</li>
</ol>
5. Elution of protein/DNA complexes
<ol>
	<li>Prepare the elution buffer (10 &#181;L of 20% SDS, 20 &#181;L of 1 M NaHCO3, 170 &#181;L of sterile distilled H2O) for all the IP and input tubes.</li>
	<li>Add 100 &#181;L of elution buffer to each centrifuge tube.</li>
	<li>Elution at 65 &#176;C for 15 min.</li>
	<li>Centrifuge for 1 min at 10,000 x g and 4 &#176;C and collect the supernatant into new centrifuge tubes.</li>
	<li>Repeat steps 5.2 - 5.4 and combine the eluates. Add 190 &#181;L of elution buffer to the 10 &#181;L of the input DNA. (total volume = 200 &#181;L).</li>
</ol>
6. Reverse crosslinking of protein/DNA complexes
<ol>
	<li>Add 8 &#181;L of 5 M NaCl to all the tubes and incubate at 65 &#176;C for 4-5 h or overnight to reverse the DNA-protein crosslinks. 
	NOTE: After this step, store the samples at -20 &#176;C, and continue the protocol the following day, if necessary.</li>
	<li>Add 1 &#181;L of RNase A and incubate for 30 min at 37 &#176;C.</li>
	<li>Add 4 &#181;L of Proteinase K (dissolved in H2O at 20 mg/mL and stored at -20 &#176;C) to each tube and incubate at 45 &#176;C for 1-2 h.</li>
</ol>
7. Purification and recovery of DNA
<ol>
	<li>Add 550 &#181;L of phenol/chloroform/isoamyl alcohol mixture (ratio of 25:24:1) to the centrifuge tube.</li>
	<li>Thoroughly vortex the mixture for 1 min.</li>
	<li>Centrifuge for 15 min at 10,000 x g and aspirate the supernatant.</li>
	<li>Transfer the extracted supernatant from the previous step to a new 1.5 mL centrifuge tube.</li>
	<li>Add 1/10 volume of 3 M sodium acetate solution, 2.5 volumes of absolute ethanol, and 3 &#181;L of glycogen (20 mg/mL) to the tube.</li>
	<li>Place the sample in a refrigerator at -20 &#176;C overnight for precipitation.</li>
	<li>Centrifuge for 15 min at 10,000 x g and 4 &#176;C.</li>
	<li>Discard the supernatant after centrifugation. Wash the pellet three times with 1 mL of 75% ethanol (needs to be prepared fresh) at 10,000 x g.</li>
	<li>Place the washed precipitate on a clean bench to let the alcohol dry.</li>
	<li>Add 50 &#181;L of sterile deionized H2O to sufficiently dissolve the precipitate.</li>
	<li>Ligate the sequencing adaptor to the DNA fragment and use a high-throughput sequencing platform to sequence the DNA.</li>
</ol>
8. DNA repair and Solexa library construction
<ol>
	<li>Repair the DNA ends to generate blunt-ended DNA using a DNA end-repair kit (1-34 &#181;L DNA, 5 &#181;L of 10x end-repair buffer, 5 &#181;L of 2.5 mM each dNTP, 5 &#181;L of 10 mM ATP, 1 &#181;L of end-repair enzyme mix, and H2O to adjust the reaction volume to 49 &#181;L).</li>
	<li>Use a PCR purification kit or phenol: chloroform extraction to purify the DNA. Elute or resuspend the DNA in 30 &#181;L of 1x TE pH 7.4.</li>
	<li>Add &#34;A&#34; to 3&#39; ends (30 &#181;L of DNA from step 2, 2 &#181;L of H2O, 5 &#181;L of 10x Taq buffer, 10 &#181;L of 1 mM dATP, and 3 &#181;L of Taq DNA polymerase). Add the reagents to a 0.2 mL PCR centrifuge tube, mix well, and react in a PCR machine at 72 &#176;C for 10 min.</li>
	<li>Perform linker ligation by mixing 10 &#181;L of DNA, 9.9 &#181;L of H2O, 2.5 &#181;L of T4 DNA ligase buffer, 0.1 &#181;L of adapter oligo mix, and 2.5 &#181;L of T4 DNA ligase. Add the reagents to a 0.2 mL PCR centrifuge tube and mix well. Incubate them at 16 &#176;C for 4 h.</li>
	<li>Purify the DNA using a PCR purification kit according to manufacturer&#39;s protocol. Elute with 20-25 &#181;L of elution buffer.</li>
	<li>Before the DNA library is established, identify the concentration of the purified DNA to confirm its usage for the subsequent sequencing experiments.</li>
	<li>Detect the DNA concentration using a fluorometer. After melting the sample on ice, mix it thoroughly and centrifuge for 30 s at 1000 x g and 4 &#176;C. Then take an appropriate amount of sample and measure it in a fluorometer with a wavelength of 260 nm.</li>
	<li>Place DNA samples with qualified quality and concentration on the Illumina sequencing platform for sequencing.</li>
	<li>Before sequencing, amplify the DNA using PCR primers, PE1.0 and PE2.0, and 2x high fidelity master mix (10.5 &#181;L of DNA, 12.5 &#181;L of 2x high fidelity master mix, 1 &#181;L of PCR primer PE1.0, and 1 &#181;L of PCR primer PE2.0). Add the reagents to a 0.2 mL PCR centrifuge tube and mix well.</li>
	<li>Run the PCR reaction in the PCR machine: 95 &#176;C predenaturation for 2 min; then 35 cycles of 95 &#176;C denaturation for 10 s, annealing at 60 &#176;C for 15 s, extension at 72 &#176;C for 5 s; a final extension at 72 &#176;C for 5 min. Finally, incubate the reaction at 4 &#176;C.</li>
	<li>Use the DNA for cluster generation and perform sequencing-by-synthesis on an Illumina Hiseq 2000. 
	NOTE: In this protocol, Illumina flow cells were used for cluster generation. The sequencing-by-synthesis was performed on an Illumina Genome Analyzer following the manufacturer&#39;s instructions.</li>
</ol>

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The authors have declared that no competing interests exist.

Recently, ChIP-seq has become a widely used genomic analysis method for determining the binding sites of TFs or enrichment sites modified by specific histones. Compared to previous ChIP-seq technology, new ChIP-seq technology is highly sensitive and flexible. Results are provided in high resolution without negative effects, such as the noise signal caused by the non-specific hybridization of nucleic acids. Although this is a common gene expression analysis, many computational methods have been validated, and the complexi...

Epigenetics is a branch of genetic research that refers to the heritable change of gene expression without changing the nucleotide sequence of genes. An increasing number of studies have shown that epigenetic regulation plays an important role in the growth and development of eukaryotic cells, including chromatin that regulates and affects gene expression through the dynamic process of folding and assembly into higher-order structures1,2. Chromatin remodeling and covalent histone modification affect and regulate the function and structure of chromatin through the variation of chromatin polymers, thereby achieving the function of regulating gene expression3,4,5,6. Posttranslational modifications of histone include acetylation, phosphorylation, methylation, monoubiquitination, sumoylation, and ADP ribosylation7,8,9. Histone H3K4 methylation, particularly trimethylation, has been mapped to transcription start sites where it is associated with transcription replication, recombination, repair, and RNA processing in eukaryotes10,11.
ChIP-seq technology was introduced in 2007 and has become the experimental standard for the genome-wide analysis of transcriptional regulation and epigenetic mechanisms12,13. This method is suitable at the genome-wide scale and for obtaining histone or transcription factor interaction information, including DNA segments of DNA binding proteins. Any DNA sequences crosslinked to proteins of interest will coprecipitate as a part of the chromatin complex. New-generation sequencing techniques are also used to sequence 36-100 bp of DNA, which are then matched to the corresponding target genome.
In phytopathogenic fungi, research has recently begun to study how histone methylation modifications regulate their target genes in the process of pathogenicity. Some previous studies proved that the regulation of histone methylase-related genes is mainly reflected in gene silencing and catalyzing the production of Secondary Metabolites (SM). MoSet1 is the H3K4 methylase in M. oryzae. Knockout of this gene results in the complete deletion of H3K4me3 modification14. Compared with the wild-type strain, the expression of the gene MoCEL7C in the mutant is inhibited in the CMC-induced state and in the non-induced state (glucose or cellobiose), the expression of MoCEL7C increased15. In Fusarium graminearum, KMT6 can catalyze the methylation modification of H3K27me3, regulate the normal development of fungi, and help regulate the &#34;cryptic genome&#34; containing the SM gene cluster16,17,18,19. In 2013, Connolly reported that H3K9 and H3K27 methylation regulates the pathogenic process of fungi through secondary metabolites and effector factors that regulate the inhibition of target genes20. In Aspergillus, the modification of histones H3K4me2 and H3K4me3 is related to gene activation and plays an important role in controlling the chromatin level regulation of SM gene clusters21. In M. oryzae, Tig1 (homologous to Tig1 in yeast and mammals) is an HADC (histone deacetylase)22. Knockout of the Tig1 gene leads to the complete loss of pathogenicity and spore production ability in the null mutant. It is more sensitive to a peroxygen environment, which cannot produce infective hyphae22.
The rice blast caused by M. oryzae. is one of the most serious rice diseases in most rice-growing areas in the world19. Due to its representative infection process, M. oryzae is similar to the infection process of many important pathogenic fungi. As it can easily carry out molecular genetic operations, the fungus has become a model organism for studying fungal-plant interactions23. Blocking every step of the infection process of M. oryzae may result in unsuccessful infection. The morphological changes during the infection process are strictly regulated by the entire genome function and gene transcription. Among them, epigenetic modifications such as histone methylation play an essential role in the transcriptional regulation of functional genes24,25. However, so far, few studies have been done on the molecular mechanism of epigenetic modifications such as histone methylation and histone acetylation in the transcription of pathogenesis genes in M. oryzae. Therefore, further developing the epigenetic regulation mechanism of the rice blast fungus while researchinh the upstream and downstream regulatory network of these pathogenic related genes will help develop rice blast prevention and control strategies.
With the development of functional genomics such as ChIP-seq, especially in epigenetics, this high-throughput data acquisition method has accelerated research on chromosomes. Using the ChIP-seq experimental technology, the genome-wide distribution of histone methylation (such as H3K4me3, H3K27me3, H3K9me3) in M. oryzae and other filamentous fungi can be identified. Therefore, this method can help elucidate the molecular mechanisms underlying how epigenetic modifications regulate their candidate target genes during fungal pathogenesis in plant pathology.

<table><tbody><tr><td>acidic casein hydrolysate</td><td>WAKO</td><td>65072-00-6</td><td>Medium configuration</td></tr><tr><td>agar powder</td><td>scientan</td><td>9002-18-0</td><td>Medium configuration</td></tr><tr><td>deoxycholic acid</td><td>MedChemExpress</td><td>83-44-3</td><td>protein and dissolution</td></tr><tr><td>DNA End-Repair kit</td><td>NovoBiotec</td><td>ER81050</td><td>Repair DNA or cDNA damaged by enzymatic or mechanical shearing</td></tr><tr><td>Dynabeads</td><td>Invitrogen</td><td>no.100.02D</td><td>Binding target</td></tr><tr><td>EB buffer</td><td>JIMEI</td><td>JC2174</td><td>Membrane and liquid</td></tr><tr><td>EDTA</td><td>ThermoFisher</td><td>AM9912</td><td>protease inhibitor</td></tr><tr><td>enzymatic casein hydrolysate</td><td>Sigma</td><td>91079-40-2</td><td>Medium configuration</td></tr><tr><td>glucose</td><td>Sigma</td><td>50-99-7</td><td>Medium configuration</td></tr><tr><td>glycogen</td><td>ThermoFisher</td><td>AM9510</td><td>Precipitant action</td></tr><tr><td>H3K4me3 antibodies</td><td>Abcam</td><td>ab8580</td><td>Immune response to H3K4me3 protein</td></tr><tr><td>illumina Genome Analyzer</td><td>illumina</td><td>illumina Hiseq 2000</td><td>Large configuration</td></tr><tr><td>Illumina PCR primers</td><td>illumina</td><td>CleanPlex</td><td>Random universal primer</td></tr><tr><td>isoamyl alcohol</td><td>chemical book</td><td>30899-19-5</td><td>Purified DNA</td></tr><tr><td>LiCl</td><td>ThermoFisher</td><td>AM9480</td><td>specific removal RNA</td></tr><tr><td>lysing enzymes</td><td>Sigma</td><td>L1412-10G</td><td>cell lysis buffer</td></tr><tr><td>Mouse IgG</td><td>Yeasen</td><td>36111ES10</td><td>Animal normal immunoglobulin</td></tr><tr><td>NaCl solution</td><td>ThermoFisher</td><td>7647-14-5</td><td>Medium configuration</td></tr><tr><td>NaHCO3</td><td>Seebio</td><td>SH30173.08*</td><td>preparation of protein complex eluent</td></tr><tr><td>NP-40</td><td>ThermoFisher</td><td>85124</td><td>cell lysate to promote cell lysis</td></tr><tr><td>PCR Purification kit</td><td>Qiagen</td><td>28004</td><td>The purification procedure removes primers from DNA samples</td></tr><tr><td>protease inhibitors</td><td>ThermoFisher</td><td>A32965</td><td>A protein inhibitor that decreases protein activity</td></tr><tr><td>Proteinase K</td><td>ThermoFisher</td><td>AM2546</td><td>DNA Extraction Reagent</td></tr><tr><td>Qubit 4.0</td><td>ThermoFisher</td><td>Q33226</td><td>Medium configuration</td></tr><tr><td>RIPA buffer</td><td>ThermoFisher</td><td>9806S</td><td>cell lysis buffer</td></tr><tr><td>RNase A</td><td>ThermoFisher</td><td>AM2271</td><td>Purified DNA</td></tr><tr><td>SDS</td><td>ThermoFisher</td><td>AM9820</td><td>cover up the charge differences</td></tr><tr><td>sodium acetate solution</td><td>ThermoFisher</td><td>R1181</td><td>Acetic acid buffer</td></tr><tr><td>sodium deoxycholate</td><td>ThermoFisher</td><td>89904</td><td>inhibition of protease degradation</td></tr><tr><td>T4 DNA ligase</td><td>ThermoFisher</td><td>EL0011</td><td>Under the condition of ATP as coenzyme, DNA ligase</td></tr><tr><td>T4 DNA ligase buffer</td><td>ThermoFisher</td><td>B69</td><td>DNA ligase buffer</td></tr><tr><td>Tris-HCl</td><td>ThermoFisher</td><td>1185-53-1</td><td>buffer action</td></tr><tr><td>Triton X-100</td><td>ThermoFisher</td><td>HFH10</td><td>keep the membrane protein stable</td></tr><tr><td>yeast extract</td><td>OXOID</td><td>LP0021</td><td>Medium configuration</td></tr></tbody></table>

genome-wide analysis of histone modifications distribution using the chromatin immunoprecipitation sequencing method in magnaporthe oryzae

Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful and widely used molecular technique for mapping whole genome locations of transcription factors (TFs), chromatin regulators, and histone modifications, as well as detecting entire genomes for uncovering TF binding patterns and histone posttranslational modifications. Chromatin-modifying activities, such as histone methylation, are often recruited to specific gene regulatory sequences, causing localized changes in chromatin structures and resulting in specific transcriptional effects. The rice blast is a devastating fungal disease on rice throughout the world and is a model system for studying fungus-plant interaction. However, the molecular mechanisms in how the histone modifications regulate their virulence genes in Magnaporthe oryzae remain elusive. More researchers need to use ChIP-seq to study how histone epigenetic modification regulates their target genes. ChIP-seq is also widely used to study the interaction between protein and DNA in animals and plants, but it is less used in the field of plant pathology and has not been well developed. In this paper, we describe the experimental process and operation method of ChIP-seq to identify the genome-wide distribution of histone methylation (such as H3K4me3) that binds to the functional target genes in M. oryzae. Here, we present a protocol to analyze the genome-wide distribution of histone modifications, which can identify new target genes in the pathogenesis of M. oryzae and other filamentous fungi.

The whole flow chart of the ChIP-seq method is shown in Figure 1. ChIP-seq experiments were performed using antibodies against H3K4me3 in the wild-type strain P131 and three null mutant strains that were devoid of mobre2, mospp1, and moswd2 gene to verify the whole genome-wide profile of histone H3K4me3 distribution in M. oryzae. The protoplasts of the wild-type strain, &#916;mobre2, &#916;mospp1, and &#916;moswd2, were prepared ...

Watch this Scientific Journal Video about Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae at JoVE.com

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

Here, we present a protocol to analyze the genome-wide distribution of histone modifications, which can identify new target genes in the pathogenesis of M. oryzae and other filamentous fungi.

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful and widely used molecular technique for mapping whole genome locations of transcription ...

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2.8K Views.  Beijing University of Agriculture. Deze methode kan helpen bij het ophelderen van de moleculaire mechanismen die ten grondslag liggen aan de regulatie van kandidaat-doelgenen via epigenetische modificaties tijdens schimmelpathogenese in plantenpathologie. Chromatine immunoprecipitatie sequencing technologie kan efficiënt DNA-segmenten detecteren die interageren met histonen en transcriptiefactoren in het hele genoom. In vergelijking met andere methoden kan het de efficiëntie en resolutie verbeteren.Begin met het toevo...

Video: Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

Environment

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors, including histone PTMs, are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for innovative bioengineering solutions. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed in their intact forms by top-down mass spectrometry (MS) coupled with online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow, thus, yielding the global PTM profile of core histones (H4, H2A, H2B, H3). We have applied this protocol previously to profile histone PTMs from sorghum leaf tissue collected from a large-scale field study, aimed at identifying epigenetic markers of drought resistance. The protocol could potentially be adapted and optimized for chromatin immunoprecipitation-sequencing (ChIP-seq), or for studying histone PTMs in similar plants.

The protocol has been developed to effectively extract intact histones from sorghum leaf materials for profiling of histone post-translational modifications that can serve as potential epigenetic markers to aid engineering drought resistant crops.

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. ...

Biochemistry

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

Biology

Visualizing Early Infection Sites of Rice Blast Disease (Magnaporthe oryzae) on Barley (Hordeum vulgare) Using a Basic Microscope and a Smartphone

Understanding how plants and pathogens interact, and whether that interaction culminates in defense or disease, is required to develop stronger and more sustainable strategies for plant health. Advances in methods that more effectively image plant-pathogen samples during infection and colonization have yielded tools such as the rice leaf sheath assay, which has been useful in monitoring infection and early colonization events between rice and the fungal pathogen, Magnaporthe oryzae. This hemi-biotrophic pathogen causes severe disease loss in rice and related monocots, including millet, rye, barley, and more recently, wheat. The leaf sheath assay, when performed correctly, yields an optically clear plant section, several layers thick, which allows researchers to perform live-cell imaging during pathogen attack or generate fixed samples stained for specific features. Detailed cellular investigations into the barley-M. oryzae interaction have lagged behind those of the rice host, in spite of the growing importance of this grain as a food source for animals and humans and as fermented beverages. Reported here is the development of a barley leaf sheath assay for intricate studies of M. oryzae interactions during the first 48 h post-inoculation. The leaf sheath assay, regardless of which species is being studied, is delicate; provided is a protocol that covers everything, from barley growth conditions and obtaining a leaf sheath, to inoculation, incubation, and imaging of the pathogen on plant leaves. This protocol can be optimized for high-throughput screening using something as simple as a smartphone for imaging purposes.

Visualizing Early Infection Sites of Rice Blast Disease Magnaporthe oryzae on Barley Hordeum vulgare Using a Basic Microscope and a Smartphone

This is a straightforward protocol of a barley leaf sheath assay using minimal reagents and common laboratory equipment (including a basic smartphone). The purpose is to visualize the early infection process of blast disease in labs without access to advanced microscopy equipment.

Understanding how plants and pathogens interact, and whether that interaction culminates in defense or disease, is required to develop stronger and more ...

Developmental Biology

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...

Neuroscience

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

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