Research Article An Ash1-Like Protein MoKMT2H Null Mutant Is …downloads.hindawi.com/journals/bmri/2016/1575430.pdf · 2019-07-30 · Research Article An Ash1-Like Protein MoKMT2H

Research ArticleAn Ash1-Like Protein MoKMT2H Null Mutant IsDelayed for Conidium Germination and Pathogenesis inMagnaporthe oryzae

Zhaojun Cao Yue Yin Xuan Sun Jun Han Qing peng SunMin Lu Jinbao Pan and Weixiang Wang

Beijing Key Laboratory of New Technique in Agricultural Application College of Plant Science and TechnologyBeijing University of Agriculture Beijing 100026 China

Correspondence should be addressed to Jinbao Pan buazkp163com and Weixiang Wang wwxbua163com

Received 2 June 2016 Revised 15 July 2016 Accepted 19 July 2016

Academic Editor Vijai Bhadauria

Copyright copy 2016 Zhaojun Cao et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Ash1 is a known H3K36-specific histone demethylase that is required for normal Hox gene expression and fertility in Drosophilaandmammals However little is known about the expression and function of the fungal ortholog of Ash1 in phytopathogenic fungusMagnaporthe oryzae Here we report that MoKMT2H an Ash1-like protein is required for conidium germination and virulencein rice We obtained MoKMT2H null mutant (ΔMoKMT2H) using a target gene replacement strategy In the ΔMoKMT2H nullmutants global histone methyltransferase modifications (H3K4me3 H3K9me3 H3K27me3 and H3K36me23) of the genomewere unaffected The ΔMoKMT2H mutants showed no defect in vegetative hyphal growth conidium morphology conidiation ordisease lesion formation on rice leaves However the MoKMT2H deletion mutants were delayed for conidium germination andconsequently had decreased virulence Taken together our results indicated that MoKMT2H plays an important role in conidiumgermination during appressorium formation in the rice blast fungus and perhaps other pathogenic plant fungi

1 Introduction

Covalent histone modifications such as methylation andacetylation provide key epigenetic information in transcrip-tional regulation and chromatin structure organization forfunctional responses [1 2] The vast array of such modifica-tions gives enormous potential for functional responses Thetiming of the appearance of amodificationwill depend on thesignaling conditions within the cell Histone methylation iscatalyzed by histonemethyltransferases (HMTs) andprovidesan excellent epigenetic mechanism for the stable transfer ofgene expression profiles to progeny cells [1 3 4]

HMTs can be grouped into twodivergent families histonelysine methyltransferases (HKMTs) catalyze the methylationof lysine residues and protein arginine methyltransferases(PRMTs) catalyze the methylation of arginine residuesHKMTs are conserved in a wide range of eukaryotes play-ing roles in cellular signaling pathways related to the cellcycle transcription and pathogenesis [1 5 6] In Fusarium

graminearum H3K4me is required for the active transcrip-tion of genes involved in deoxynivalenol and aurofusarinbiosynthesis [7]ThehistoneH3K4methyltransferase FgSET1plays important roles in the response to agents that damagethe cell wall by negatively regulating the phosphorylationof FgMgv1 [7 8] Another H3K27me3 methyltransferaseKMT6 regulates the development and expression of sec-ondary metabolite gene clusters in F graminearum [9]

The SET domain is an evolutionarily conserved domainfound in HMTs In Neurospora crassa the SET domain-containing protein set-2 encodes a histone H3K36 methyl-transferase and is essential for normal growth and develop-ment [10ndash12] The Drosophila trithorax group protein absentsmall or homeotic discs 1 (Ash1) contains a SET domain andis involved in maintaining active transcription of many genesby conferring a relaxed chromatin structure [13ndash20] Inmam-mals Ash1-like (ASH1L) is the Ash1 homolog and occupiesmost actively transcribed genes andmethylates histone H3 ina nonredundant fashion at a subset of genes including Hox

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 1575430 9 pageshttpdxdoiorg10115520161575430

2 BioMed Research International

Table 1 Fungal strains used in this study

Strain Description ReferenceP131 The wild-type strain Peng and Shishiyama (1988) [29]KO1 The deletion mutant ofMoKMT2H This studyKO2 The deletion mutant ofMoKMT2H This studyKO3 The deletion mutant ofMoKMT2H This study

genes [13 17 20 21] In human HeLa cells Ash1 methylateshistone H3 at lysine 36 and regulates the development ofmyelomonocytic lineages fromhematopoietic stem cells [22]However roles for the fungal Ash1 ortholog a well-studiedH3K36me2 methyltransferase have not yet been extensivelyexplored inM oryzae

M oryzae is a causal agent of rice blast disease whichis the most devastating and persistent disease in cultivatedrice [23 24] M oryzae can produce a three-celled asexualspore called a conidium as its primary source of inoculumWhen a conidium lands on the surface of a leaf from a riceplant it germinates to form an appressorium as the infectionstructureThe appressorium can enter into the host plant andgrow inside the plant tissues leading to yield losses or evendeath of the infected plant [25ndash28] Recently several studieshave focused on the molecular mechanism of HKMTs inplant pathogenesis and plant infection [7 8 29] The Moryzae histone H3K4me3 methyltransferase gene SET1 wastargeted for gene disruption by homologous recombina-tion [28] Chromatin immunoprecipitation-sequence analy-sis indicated that MoSET1 can regulate global gene expres-sion during infection-related morphogenesis [28] Widelyconserved MoKMT2H in ascomycete fungi is a functionalhomolog of Ash1 which is implicated in H3K4 and H3K36methylation In a previous study researchers used a wheat-infecting strain to confirm that the MoKMT2H gene isinvolved in infection-related morthogenesis and pathogenic-ity to wheat and barley cultivars other than rice [28] Here wetargeted the Ash1-likeMoKMT2H gene for gene replacementto further determine the function of MoKMT2H in a rice-infecting M oryzae strain The ΔMoKMT2H null mutanthas no obvious defect in vegetative hyphal growth conidiummorphology conidiation or disease lesion formation on riceand wheat leaves However the ΔMoKMT2H null mutantwas delayed for conidium germination and consequentlyhad decreased virulence This study offers evidence for theinvolvement of the histone methyltransferase function ofAshl-like proteins during plant pathogenesis andmayprovideimportant implications for discovering new HKMTs in therice blast fungus and perhaps other pathogenic plant fungi

2 Materials and Methods

21 Antibodies Antibodies against histone H3 (Abcamab1791) H3K36me3 (Abcam ab9095) H3K36me2 (Abcamab9049) H3K4me3 (Abcam ab8580) H3K27me3 (Upstate07-449) and H3K9me3 (Millipore 07-442) were purchasedcommercially

22 Fungal Strains and Growth Conditions The wild-typestrain P131 ofMagnaporthe oryzaewas used in this study P131was the field isolate [29] which was used to generate mutantstrains The wild-type strain P131 and all the transformantstrains generated in this studywere listed inTable 1Theyweregrown on oatmeal tomato agar (OTA) medium and culturedat 25∘C under light conditions [29ndash31] For extraction ofgenomic DNA and protein and isolation of protoplasts freshmycelia were braked and cultured in liquid completemedium(CM 06 yeast extract 03 enzymatic casein hydrolysate03 acidic casein hydrolysate and 1 glucose) and shakenat 150 rpm at 25∘C for 36 h To determine fungal growthmycelial plugs of sim5mm in diameter were inoculated onOTA plates Colony diameter on each plate was measured forsim3 days Each experiment was repeated three times

23 Molecular Manipulations with Nucleic Acids For gen-omic DNA extraction genomic DNA was isolated using thecetyltrimethylammonium bromide protocol as described [3132] sim1 to 2 g mycelia was harvested and ground into a finepowder and then extracted with 15mL of extraction buffer(50mM Tris-HCl 100mM EDTA and 150mM NaCl) Add750120583L 20 SDS mix well and incubate at 37∘C for 1 h Add225mL 5MNaCl and 2mL CTABNaCl buffer (10 CTAB07M NaCl) mix well and then incubate at 65∘C for 30minAdd chloroform isoamyl alcohol (24 1) and mix well Celldebris was removed by centrifugation at 10000 rpm for15min and genomic DNA was pelleted by isopropyl alcoholThe pellet was washed with 70 alcohol and dissolved insterilized-distilled water and stored in minus20∘C For Southernblot analysis the extracted genomic DNA was completelydigested by the restriction enzyme EcoRI digestion andagarose gel separation and DNA gel blotting were performedfollowing the standard protocols [29 30 32] Hybridizationwas performed in solution containing 6x SSC 5x Denhardtrsquossolution 05 SDS and 100 120583LmLminus1 denatured salmonsperm DNA at 65∘C Blots were exposed to phosphorimageranalyzer (BAS-2040 Fuji Photo Film Co Ltd Tokyo Japan)and visualized by phosphorimager analyzer software

24 Generation of the MoKMT2H Gene Replacement Strainswith Split-PCR Strategy For generating theMoKMT2H genereplacement stains target gene replacement was carriedout using the split-PCR strategy [33] The primers used toamplify the flanking sequences from the genomic DNA ofP131 in this study are listed in Table 2 For the first roundof PCR the upstream and downstream flanking sequences

BioMed Research International 3

Table 2 PCR primers used in this study

Primer Sequence (51015840-31015840)LBCK GCCTGTCTGATTGGARBCK GGGAGGGGGTGATGACGGTCLB-F CTGCTGCTTAGGTCGGTAGTCTLB-R TTGACCTCCACTAGCTCCAGCCAAGCCATTGGCTGGTTGGTTTGTTGGTRB-F GAATAGAGTAGATGCCGACCGGGCGTCACATGCGAACAAGAACCARB-R GGCAAGGCAAGATTGGCTAAGAHYG-F CTTGGCTGGAGCTAGTGGAGGTHYG-R CCCGGTCGGCATCTACTCTATTCHYG-F1 CGTTGCAAGACCTGCCTGAAHYG-R1 GGATGCCTCCGCTCGAAGTAHYG-LBCK GACAGACGTCGCGGTGAGTTHYG-RBCK TCTGGACCGATGGCTGTGTAG

UPF GAGAACTCAA GCGTCACTCCUPR GAACCAAAAGCATGTTTCTPr2937F CCTTGCCTGTCTGATTGGPr2937R GGAGTGACGCTTGAGTTC

were amplified with the primer pairs LBCKLB-R and RB-FRBCK respectively For the second roundof PCR the fused2937-HYG DNA flanking fragments of the left arm and rightarmwere amplified from theDNAproducts of the first-roundPCR with the primers LB-FHYG-R1 and RB-RHYG-F1respectively Protoplasts were isolated and the two flankingsequences were transformed into P131 protoplasts using theclassic method [32 33] For selecting hygromycin-resistantor neomycin-resistant transformants CM plates were sup-plemented with 250120583gmLminus1 hygromycin B (Roche USA)or 400 120583gmLminus1 neomycin (Ameresco USA) The selectedneomycin-resistant transformants were subjected to PCRwith primers UPFHYG-R and UPRHYG-F respectivelyand Southern blot analysis

25 Protein Extraction and Western Blot Analysis For all teststrains fresh mycelia were disturbed finely and transferredto liquid CM and shaken at 150 rpm at 25∘C for 36 hThe resulting mycelia (sim15 g) were harvested and frozen inliquid nitrogen The frozen mycelia were ground into a finepowder and transferred to 10mL of protein extraction buffer[50mM Tris-HCl pH 75 100mM NaCl 5mM ethylenediamine tetraacetic acid (EDTA) 1 triton X-100 and 2mMphenylmethylsulfonyl fluoride (PMSF)] and 10 120583L of proteaseinhibitor cocktail (Roche shanghai China) as described [8]After homogenization with a vortex shaker the resultinglysate was centrifuged at 12000timesg for 30min at 4∘C Thesupernatant was then separated by 13 denaturing polyacry-lamide gel (SDS-PAGE) transferred to Immobilon-P transfermembrane (Millipore Billerica MA USA) with a Bio-Radelectroblotting apparatus and evaluated using the antibod-ies anti-histone H3 anti-H3K36me3 anti-H3K36me2 anti-H3K4me3 anti-H3K27me3 and anti-H3K9me3 respectively[8] Incubation with a secondary antibody (Santa Cruz CAUSA) and chemiluminescent detection were performed asdescribed previously [8 29]

26 Plant Virulence Test and Observation of Infection Forthe infection assayM oryzae conidia were collected from 7-day-old OTA plates and resuspended to sim2 times 104 conidiamLin 0025 Tween-20 solution Four-week-old seedlings ofrice (Oryzae sativa) cv Lijiangxintuanheigu and 8-day-oldseedlings of barley (Hordeum vulgare) cv E9 were usedfor spray infection assays [29 30] Plant incubation wasperformed as described previously [29 30] Conidia weresprayed onto the barley or rice leaves which were thenincubated in a moist and dark chamber at 28∘C At 9 h and36 h after inoculation the leaf samples were observed bymicroscopy Lesion formation was examined 7 days afterinoculation For mycelium plug inoculation the myceliumplugs of the ΔMoKMT2H null mutants and the wild-typestrain were inoculated onto the abraded rice plants leavesand barley leaves For conidia droplet inoculation conidiasuspended to sim2 times 105 conidiamL were inoculated ontothe abraded rice plants leaves Lesions were examined andphotographed 5ndash7 days after inoculation The mean numberof lesions formed on 5 cm leaf tips was determined asdescribed previously [29 30]

3 Results

31 Target Gene Replacement of MoKMT2H in M oryzaeMoKMT2H is widely conserved in ascomycete fungiMoKMT2H is similar to mammalian Ash1l protein which isrequired for H3K36me2methylation In previous studies theΔMoKMT2H mutants showed a significant reduction in veg-etative growth germination appressorium formation andpathogenicity to host plants [25] However the researchersused a wheat-infectingM oryzae strain in their experimentsand their testedM oryzae strain cannot infect rice To furtherdetermine the function of MoKMT2H in a rice-infectingM oryzae strain we performed target gene replacement ofMoKMT2H with a hygromycin phosphotransferase cassette


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Figure 1 The strategy for MoKMT2H gene replacement with PCR (a) Schematic representation of the genomic DNA of MoKMT2H Blueboxes indicate exons The ATG start codon and TAA stop codon are indicated The EcoRI restriction enzyme sites are indicated The red linelabeled ldquoproberdquo shows the region used for Southern blot analysis The upstream and downstream flanking sequences were amplified withprimer pairs LBCKLB-R and RBCKRB-F respectively The fused DNA fragments with HYG were amplified with LB-FHYG-R1 and HYG-F1RB-R The DNA fragments used for transformants were amplified with LB-FRB-F (b) Southern blot analysis of EcoRI-digested genomicDNA from wild-type P131 and the neomycin-resistant transformants KO1 KO2 and KO3 Blots were hybridized with probe as indicatedin (a) The DNA fragment used for a probe was amplified with primer pair Pr2937FPr2937R (c) The transformants KO2 and KO3 wereconfirmed by amplification with primer pairs UPFHYG-R and UPRHYG-F

using the split-marker recombination method (Figure 1(a))Three neomycin-resistant transformants KO1 KO2 andKO3 were selected for verification by PCR or Southern blotanalysis To further characterize the MoKMT2H gene dele-tion in the selected strains KO1 KO2 andKO3 we performedSouthern blot analysis using a DNA fragment as a probeThe whole genomes of KO1 KO2 and KO3 were digested

with EcoRI When probed with a 03-kb DNA fragmentamplified with primer pairs Pr2937FPr2937R (Table 2) theΔMoKMT2H null mutants produced a 39-kp band whereasthe wild-type strain had a 65-kp band (Figure 1(b)) ForPCR verification we used two primers UPFHYG-R andHYG-FUPR in our study When amplified with primerpair UPFHYG-R the ΔMoKMT2H but not wild-type P131


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Figure 2 Comparisons of colonymorphology hyphal growth rate and conidiation capacity of wild type (P131) and the gene deletionmutantsof MoKMT2H (a) Colony morphology of each strain incubated on OTA plates for 5 days at 25∘C (b) Colony diameter of each strain (c)Theaverage number of conidia for each strain Values are the mean plusmn SD from three biological replicates

strain had a 26-kb DNA band whereas the ΔMoKMT2Hstrain had a 11-kb DNA band with primers HYG-FUPR(Figure 1(c)) These results confirm that theMoKMT2H genewas completely replaced

32 MoKMT2H Is Required for Conidium Formation andPathogenicity on Wound Leaves of Rice To further charac-terize the biological and chemical roles of MoKMT2H in Moryzae we compared the colony morphology hyphal growthrate conidiation capacity and virulence on the leaves ofrice The ΔMoKMT2H null mutants and wild-type P131 wereincubated on OTA plates for 3 days The ΔMoKMT2H nullmutants showed no obvious defects in colony morphology(Figure 2(a)) hyphal growth rate (Figure 2(b)) or thecapacity for conidium formation (Figure 2(c)) in comparisonwith the wild-type P131 strain (Figure 2(a)) suggesting thatMoKMT2H is not required for asexual development inM Oryzae The ΔMoKMT2H null mutant strain KO3 wasselected for plant virulence test To evaluate virulence ofthe ΔMoKMT2H null mutants conidia suspensions from

the ΔMoKMT2H null mutants and from wild-type P131were inoculated onto the surface of barley The ΔMoKMT2Hconidium formation was reduced by 61 in comparison withthe wild type 9 h after inoculation (Figures 3(a) and 3(b))Strikingly at 36 h after inoculation wild-type P131 formedbulbous infection hyphae and could extend to neighboringhost cells whereas the ΔMoKMT2H null mutants had obvi-ous defects in penetration peg formation (Figure 3(a)) Tofurther confirm the plant infection defects in ΔMoKMT2Hnull mutants conidia suspensions of the ΔMoKMT2H nullmutants and wild-type P131 were sprayed onto seedlings ofrice cultivar LTH Typical robust lesions of the rice blastwere observed for the wild type However the lesions ofΔMoKMT2H null mutants were not markedly reduced ascompared with the wild type (Figure 3(c)) For the barleyleaves typical disease lesions were observed from both thewild type and the ΔMoKMT2H null mutant strains follow-ing spray inoculation of conidia (Figure 3(d)) To furthercheck whether the ΔMoKMT2H null mutants could infectthe host cells through wounds the mycelium plugs of theΔMoKMT2H null mutants and wild type were inoculated


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ΔMoKMT2H P131 ΔMoKMT2H P131 ΔMoKMT2H

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(d)

Figure 3 MoKMT2H is required for conidium formation and pathogenicity on rice leaves (a) Microscopic observation of conidiumformation in thewild-type strain andΔMoKMT2Hnullmutants on barley leaves at 9 and 36 h after inoculation Bar 20 120583mAP appressoriumIH infectious hyphae (b) Conidiation of P131 and ΔMoKMT2H null mutants after growth on OTA plates for 7 days Comparison of theconidium formation rate betweenP131 andΔMoKMT2Hnullmutants at 48 h after conidiationValues are themeanplusmn SD from three biologicalreplicates (c) Conidia spray (left) mycelium plug (middle) and conidia droplet inoculation on rice leaves with conidia from the wild-typeP131 and the ΔMoKMT2H null mutants Typical leaves were observed 7 days after mycelium plug inoculation Mycelium plug and conidiadroplet inoculation was conducted on abraded rice leaves Typical leaves were observed 5 days after mycelium plug inoculation (d) Conidiaspray (left) and mycelium plug (right) inoculation on barley leaves with conidia from the wild-type P131 and the ΔMoKMT2H null mutantsf indicates 119875 lt 005

onto abraded rice leaves (Figure 3(c)) Although inoculationwith the wild-type strain caused severe and typical lesionson wound rice leaves the ΔMoKMT2H null mutants couldnot cause any disease lesions suggesting that MoKMT2Hwas essential for penetration peg formation on wounded riceleaves (Figure 3(c)) To further confirm our observation weperformed conidia droplet inoculation onwounded rice plantleaves As shown in Figure 3(c) inoculation with the wild-type caused typical disease lesions around the wound sitescompared with the ΔMoKMT2H null mutants Thereforethese results suggest that MoKMT2H regulates conidiumformation and penetration peg formation in wound patho-genesis on rice leaves

33 MoKMT2H Is a Conserved Set Domain-Containing Pro-tein but Is Not Involved in Genome-Wide Histone MethylationThe SET domain which mediates lysine methylation regu-lates chromatin-mediated gene transcription Published stud-ies show that SETproteins in lower organisms canmanipulatehost transcription machinery in host-pathogen interactions[10 11] MoKMT2H is an Ashl-like protein [12] Ash1-likeorthologs ofMoKMT2Hare also present inDrosophilaHomosapiens Mus musculus F graminearum and M oryzae [10ndash16] To further identify the homologs of Ash1-like proteinswe assessed the conservation of the SET domains throughamino acid sequence alignment (Figure 4) The sequencealignment in Figure 4 highlights the conservation of the


L R FR AEEK W I TKEPLKAGQF I L V VS Q FR N


V R FM TA DK W V TKLPIA KGTY L V V VT K FKQ

V V IKTSDR Y V SNR CFR PNQI M A IIT D CER

V V VHTGPR F V ASR CFEPGQI M A IIT E CERe

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ASI LNDTHH CLHL GGLV I GQR M SDC V E

TEV KDNECY LM SF QNM II A TT SIA V N

NEV KDNEA Y LM SF QNM IL A TT SIA V S

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Homo sapiensMus musculusDrosophila melanogasterFusarium graminearumMagnaporthe oryzae

SET domain

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Figure 4Multiple alignment of SETdomain ofAshl-like proteins in classic speciesThebackground colors indicate the conservation of aminoacid residues Orange shading color represents ge50 similarity green represents ge75 similarity and black represents 100 similarity Theconserved residues are indicated by red lines the SET domain residues in the NHxxxPN motif and the post-SET domain which consists ofthe cysteine-rich motif CxCxxxxC that binds Zn2+ OR that coordinates Zn2+ binding are indicated

SET domain residues in the NHxxxPN motif and the post-SET domain containing the Zn2+-bindingmotif (CxCxxxxC)among these selected Ash1-like proteins Collectively thesedata indicate that MoKMT2H is highly conserved acrosslower eukaryotes to higher animals which suggests thatthese SET domain-containing proteins may have evolvedimportant roles in manipulating chromatin methylation-mediated gene transcription

Considering that MoKMT2H is a candidate histonemethyltransferase we decided to apply western blot analysisto determine whether the methylation pattern of genome-wide histone lysines in the ΔMoKMT2H null mutants waschanged Some groups reported that MoKMT2H is notspecific for H3K4 H3K27me3 or H3K20me3 methylationin their KMT null mutants [28] However some groupsproved that the SET domain of MoKMT2H is much moreclosely related to H3K36me2-specific methyltransferasesthan H3K4-specific methyltransferases such as Set1 or tritho-rax group proteins [20 22] Total proteins were extractedfrom the mycelia of ΔMoKMT2H null mutant and wild-typestrains There was no significant reduction in the signal ofH3K4me3 H3K9me3 H3K27me3 and H3K36me3 methyla-tion in the ΔMoKMT2H null mutant (Figure 5) Moreoverthe ΔMoKMT2H null strain also displayed no activity onH3K36me2 of the genome-wide chromatin It is possible

that MoKMT2H may act only on some specific target genesduring the process of pathogenesis

4 Discussion

The SET domain which mediates lysine methylation is oneof the major epigenetic marks and regulates chromatin-mediated gene transcription [10 12 17] The multiple align-ment depicts the evolutionary history of the conserved SETdomain of MoKMT2H an Ash1-like protein The presenceof the cysteine-rich (CxCxxxxC) post-SET domain facili-tates the binding of Zn2+ ions and marks an evolution-ary divergence from the homologs in other organisms Inaddition the 119878-adenosylmethionine cofactor-binding siteand the post-SET motif-containing region are also highlyconserved In our study we did not however detect a changein genome-wide histone methylation on H3K36me23H3K4me3 H3K9me3 or H3K27me3 in the ΔMoKMT2Hnull strains (Figure 5 and data now shown) which suggeststhat MoKMT2H may have methylation activity only onspecific target genes of histone H3 or other key regulatedproteins during the process of plant pathogenesis Furtherexperiments will focus on the HMT activity on recombinantnucleosomes or core histones with recombinant MoKMT2HSET domain deletions in vitro


H3K4me3

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H3K36me2

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P131 ΔMoKMT2H

Figure 5 Western blot analysis of multiple histone modificationsin the wild type and ΔMoKMT2H null mutants Total proteinswere extracted and separated by 13 SDS-PAGE followed byimmunoblotting with the indicated antibodies Anti-H3 was usedas a loading control

MoKMT2H is required for conidiation conidium ger-mination and appressorium formation [26 28 29] andΔMoKMT2H null strains are defective for pathogenicity onwheat cultivar Norin 4 [28] However those previously testedwild-type and null mutant strains can only infect wheat Tofurther determine the biological and chemical activity ofMoKMT2H we generated ΔMoKMT2H null strains fromthe P131 wild-type strain which forms classical lesions onrice leaves The most interesting conclusion in this studywe obtained when compared with previous studies is thatdeletion of MoKMT2H reduced the rate of conidium for-mation and plant virulence on wounded rice leaves Moreinterestingly spray inoculation of the ΔMoKMT2H nullstrains did not cause any reduction in pathogenicity onrice leaves (Figures 3(c) and 3(d)) These results suggestthat MoKMT2H may recognize specific effectors induced bywounds during the plant-pathogen interaction Our studyhas thus shown that the Ash1-like protein MoKMT2H isrequired for conidium germination and pathogenesis in Moryzae MoKMT2H is widely conserved in ascomycete fungihowever its biological roles have not been uncovered Furtherresearch will focus on determining whether MoKMT2H has

the chemical activity of a histone methyltransferase andisolating the proteins that it interacts with and its downstreamtarget genes during pathogenesis

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Zhaojun Cao and Yue Yin contributed equally to this workWeixiang Wang designed research analyzed data and wrotethe paper Zhaojun Cao and Yue Yin performed researchMin Lu Qing peng Sun JunHan and Jinbao Pan contributedanalytic tools

Acknowledgments

The authors thank Dr Youliang Peng at China AgriculturalUniversity for providing the wild-type strain P131 andΔMoKMT2H transformed strains of M oryzae Thiswork was supported by Special Scientific Research Projectof Beijing Agriculture University (YQ201603) and theScientific Project of Beijing Educational Committee(KM201610020005)

References

[1] T Kouzarides ldquoChromatin modifications and their functionrdquoCell vol 128 no 4 pp 693ndash705 2007

[2] R Margueron P Trojer and D Reinberg ldquoThe key to devel-opment interpreting the histone coderdquo Current Opinion inGenetics and Development vol 15 no 2 pp 163ndash176 2005

[3] M Grunstein ldquoHistone acetylation in chromatin structure andtranscriptionrdquo Nature vol 389 no 6649 pp 349ndash352 1997

[4] CMartin andY Zhang ldquoThediverse functions of histone lysinemethylationrdquo Nature Reviews Molecular Cell Biology vol 6 no11 pp 838ndash849 2005

[5] J C Black C Van Rechem and J R Whetstine ldquoHistonelysine methylation dynamics establishment regulation andbiological impactrdquo Molecular Cell vol 48 no 4 pp 491ndash5072012

[6] E L Greer and Y Shi ldquoHistone methylation a dynamic markin health disease and inheritancerdquoNature ReviewsGenetics vol13 no 5 pp 343ndash357 2012

[7] J Jeon J Choi G-W Lee et al ldquoGenome-wide profiling ofDNAmethylation provides insights into epigenetic regulation offungal development in a plant pathogenic fungusMagnaportheoryzaerdquo Scientific Reports vol 5 article 8567 2015

[8] Y Liu N Liu Y Yin Y Chen J Jiang and Z Ma ldquoHis-tone H3K4 methylation regulates hyphal growth secondarymetabolism and multiple stress responses in Fusarium gramin-earumrdquo Environmental Microbiology vol 17 no 11 pp 4615ndash4630 2015

[9] L R Connolly K M Smith and M Freitag ldquoThe Fusariumgraminearum histone H3 K27 methyltransferase KMT6 Regu-lates development and expression of secondary metabolite geneclustersrdquo PLoS Genetics vol 9 no 10 Article ID e1003916 2013


[10] C Qian and M-M Zhou ldquoSET domain protein lysine methyl-transferases structure specificity and catalysisrdquo Cellular andMolecular Life Sciences vol 63 no 23 pp 2755ndash2763 2006

[11] A J Bannister R Schneider and T Kouzarides ldquoHistonemethylation dynamic or staticrdquo Cell vol 109 no 7 pp 801ndash806 2002

[12] C Nwasike S Ewert S Jovanovic S Haider and S MujtabaldquoSET domain-mediated lysine methylation in lower organismsregulates growth and transcription in hostsrdquo Annals of the NewYork Academy of Sciences 2016

[13] K N Byrd and A Shearn ldquoASH1 a Drosophila trithorax groupprotein is required for methylation of lysine 4 residues onhistone H3rdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 100 no 20 pp 11535ndash115402003

[14] G D Gregory C R Vakoc T Rozovskaia et al ldquoMammalianASH1L is a histone methyltransferase that occupies the tran-scribed region of active genesrdquo Molecular and Cellular Biologyvol 27 no 24 pp 8466ndash8479 2007

[15] A Shearn ldquoThe ash-1 ash-2 and trithorax genes of Drosophilamelanogaster are functionally relatedrdquo Genetics vol 121 no 3pp 517ndash525 1989

[16] T Klymenkoand and J Muller ldquoThe histone methyltransferasesTrithorax and Ash1 prevent transcriptional silencing by Poly-comb group proteinsrdquo EMBO Reports vol 5 no 4 pp 373ndash3772004

[17] B Papp and J Muller ldquoHistone trimethylation and the main-tenance of transcriptional ON and OFF states by trxG and PcGproteinsrdquoGenes andDevelopment vol 20 no 15 pp 2041ndash20542006

[18] Y B Schwartz T G Kahn P Stenberg K Ohno R Bourgonand V Pirrotta ldquoAlternative epigenetic chromatin states ofpolycomb target genesrdquo PLoS Genetics vol 6 no 1 Article IDe1000805 2010

[19] C Beisel A Imhof J Greene E Kremmer and F Sauer ldquoHis-tone methylation by the Drosophila epigenetic transcriptionalregulator Ash1rdquo Nature vol 419 no 6909 pp 857ndash862 2002

[20] Y Tanaka Z-I Katagiri K Kawahashi D Kioussis and SKitajima ldquoTrithorax-group protein ASH1 methylates histoneH3 lysine 36rdquo Gene vol 397 no 1-2 pp 161ndash168 2007

[21] Y B Schwartz T G Kahn P Stenberg K Ohno R Bourgonand V Pirrotta ldquoAlternative epigenetic chromatin states ofpolycomb target genesrdquo PLoS Genetics vol 6 no 1 articlee1000805 2010

[22] W YuanM Xu C Huang N Liu S Chen and B Zhu ldquoH3K36methylation antagonizes PRC2-mediated H3K27 methylationrdquoThe Journal of Biological Chemistry vol 286 no 10 pp 7983ndash7989 2011

[23] R A Wilson and N J Talbot ldquoUnder pressure investigatingthe biology of plant infection by Magnaporthe oryzaerdquo NatureReviews Microbiology vol 7 no 3 pp 185ndash195 2009

[24] D G O Saunders S J Aves and N J Talbot ldquoCell cycle-mediated regulation of plant infection by the rice blast fungusrdquoPlant Cell vol 22 no 2 pp 497ndash507 2010

[25] P Skamnioti and S J Gurr ldquoAgainst the grain safeguarding ricefrom rice blast diseaserdquo Trends in Biotechnology vol 27 no 3pp 141ndash150 2009

[26] N J Talbot ldquoOn the trail of a cereal killer exploring the biologyofMagnaporthe griseardquo Annual Review of Microbiology vol 57pp 177ndash202 2003

[27] M J Gilbert C R Thornton G E Wakley and N J Talbot ldquoAP-type ATPase required for rice blast disease and induction ofhost resistancerdquo Nature vol 440 no 7083 pp 535ndash539 2006

[28] K T Minh Pham Y Inoue B Van Vu et al ldquoMoSET1 (His-tone H3K4methyltransferase inMagnaporthe oryzae) regulatesglobal gene expression during Infection-related morphogene-sisrdquo PLOS Genetics vol 11 no 7 Article ID e1005385 2015

[29] Y L Peng and J Shishiyama ldquoTemporal sequence of cytologicalevents in rice leaves infected with Pyricularia oryzaerdquoCanadianJournal of Botany vol 66 no 4 pp 730ndash735 1988

[30] J Yang L Kong X Chen et al ldquoA carnitine-Acylcarnitinecarrier protein MoCrc1 is essential for pathogenicity in Mag-naporthe oryzaerdquo Current Genetics vol 58 no 3 pp 139ndash1482012

[31] J-R Xu and J E Hamer ldquoMAP kinase and cAMP signalingregulate infection structure formation and pathogenic growthin the rice blast fungus Magnaporthe griseardquo Genes and Devel-opment vol 10 no 21 pp 2696ndash2706 1996

[32] J Yang X Zhao J Sun et al ldquoA novel protein com1 isrequired for normal conidium morphology and full virulencein Magnaporthe oryzaerdquo Molecular Plant-Microbe Interactionsvol 23 no 1 pp 112ndash123 2010

[33] R S Goswami ldquoTargeted gene replacement in fungi using asplit-marker approachrdquo inPlant Fungal PathogensMethods andProtocols M D Bolton and B P H JThomma Eds vol 835 ofMethods in Molecular Biology pp 255ndash269 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Table 1 Fungal strains used in this study

Strain Description ReferenceP131 The wild-type strain Peng and Shishiyama (1988) [29]KO1 The deletion mutant ofMoKMT2H This studyKO2 The deletion mutant ofMoKMT2H This studyKO3 The deletion mutant ofMoKMT2H This study

genes [13 17 20 21] In human HeLa cells Ash1 methylateshistone H3 at lysine 36 and regulates the development ofmyelomonocytic lineages fromhematopoietic stem cells [22]However roles for the fungal Ash1 ortholog a well-studiedH3K36me2 methyltransferase have not yet been extensivelyexplored inM oryzae

M oryzae is a causal agent of rice blast disease whichis the most devastating and persistent disease in cultivatedrice [23 24] M oryzae can produce a three-celled asexualspore called a conidium as its primary source of inoculumWhen a conidium lands on the surface of a leaf from a riceplant it germinates to form an appressorium as the infectionstructureThe appressorium can enter into the host plant andgrow inside the plant tissues leading to yield losses or evendeath of the infected plant [25ndash28] Recently several studieshave focused on the molecular mechanism of HKMTs inplant pathogenesis and plant infection [7 8 29] The Moryzae histone H3K4me3 methyltransferase gene SET1 wastargeted for gene disruption by homologous recombina-tion [28] Chromatin immunoprecipitation-sequence analy-sis indicated that MoSET1 can regulate global gene expres-sion during infection-related morphogenesis [28] Widelyconserved MoKMT2H in ascomycete fungi is a functionalhomolog of Ash1 which is implicated in H3K4 and H3K36methylation In a previous study researchers used a wheat-infecting strain to confirm that the MoKMT2H gene isinvolved in infection-related morthogenesis and pathogenic-ity to wheat and barley cultivars other than rice [28] Here wetargeted the Ash1-likeMoKMT2H gene for gene replacementto further determine the function of MoKMT2H in a rice-infecting M oryzae strain The ΔMoKMT2H null mutanthas no obvious defect in vegetative hyphal growth conidiummorphology conidiation or disease lesion formation on riceand wheat leaves However the ΔMoKMT2H null mutantwas delayed for conidium germination and consequentlyhad decreased virulence This study offers evidence for theinvolvement of the histone methyltransferase function ofAshl-like proteins during plant pathogenesis andmayprovideimportant implications for discovering new HKMTs in therice blast fungus and perhaps other pathogenic plant fungi

2 Materials and Methods

21 Antibodies Antibodies against histone H3 (Abcamab1791) H3K36me3 (Abcam ab9095) H3K36me2 (Abcamab9049) H3K4me3 (Abcam ab8580) H3K27me3 (Upstate07-449) and H3K9me3 (Millipore 07-442) were purchasedcommercially

22 Fungal Strains and Growth Conditions The wild-typestrain P131 ofMagnaporthe oryzaewas used in this study P131was the field isolate [29] which was used to generate mutantstrains The wild-type strain P131 and all the transformantstrains generated in this studywere listed inTable 1Theyweregrown on oatmeal tomato agar (OTA) medium and culturedat 25∘C under light conditions [29ndash31] For extraction ofgenomic DNA and protein and isolation of protoplasts freshmycelia were braked and cultured in liquid completemedium(CM 06 yeast extract 03 enzymatic casein hydrolysate03 acidic casein hydrolysate and 1 glucose) and shakenat 150 rpm at 25∘C for 36 h To determine fungal growthmycelial plugs of sim5mm in diameter were inoculated onOTA plates Colony diameter on each plate was measured forsim3 days Each experiment was repeated three times

23 Molecular Manipulations with Nucleic Acids For gen-omic DNA extraction genomic DNA was isolated using thecetyltrimethylammonium bromide protocol as described [3132] sim1 to 2 g mycelia was harvested and ground into a finepowder and then extracted with 15mL of extraction buffer(50mM Tris-HCl 100mM EDTA and 150mM NaCl) Add750120583L 20 SDS mix well and incubate at 37∘C for 1 h Add225mL 5MNaCl and 2mL CTABNaCl buffer (10 CTAB07M NaCl) mix well and then incubate at 65∘C for 30minAdd chloroform isoamyl alcohol (24 1) and mix well Celldebris was removed by centrifugation at 10000 rpm for15min and genomic DNA was pelleted by isopropyl alcoholThe pellet was washed with 70 alcohol and dissolved insterilized-distilled water and stored in minus20∘C For Southernblot analysis the extracted genomic DNA was completelydigested by the restriction enzyme EcoRI digestion andagarose gel separation and DNA gel blotting were performedfollowing the standard protocols [29 30 32] Hybridizationwas performed in solution containing 6x SSC 5x Denhardtrsquossolution 05 SDS and 100 120583LmLminus1 denatured salmonsperm DNA at 65∘C Blots were exposed to phosphorimageranalyzer (BAS-2040 Fuji Photo Film Co Ltd Tokyo Japan)and visualized by phosphorimager analyzer software

24 Generation of the MoKMT2H Gene Replacement Strainswith Split-PCR Strategy For generating theMoKMT2H genereplacement stains target gene replacement was carriedout using the split-PCR strategy [33] The primers used toamplify the flanking sequences from the genomic DNA ofP131 in this study are listed in Table 2 For the first roundof PCR the upstream and downstream flanking sequences








3 Results



HYG-LBCK

ATG TAA

LBCK

LB-R

HYG

HYG-R1

Probe

EcoRI EcoRI

Genomic DNA

HYG-R

LB-F

HYG-F

RB-R RBCK

HYG-RRB-F

HY

YG

HYG-F1

LB

RB

LB

LBCK

RBCKUPRUPF

HYG RB

HYG-RBCK

MoKMT2H

400 bp

(a)

39 Kb

65 Kb

231 Kb

94 Kb

65 Kb

43 Kb

23 Kb20 Kb

KO3KO2KO1P131Marker

(b)

11 Kb

26 KbUPF+HYG-R

UPR+HYG-F

P131 KO2 KO3

(c)





P131 KO2 KO3

(a)

50

100

150

200

250

300

350

400

450

Col

ony

diam

eter

(mm

)

KO2 KO3P131(b)

10

20

30

40

50

60

Num

ber o

f con

idia

(107)

KO2 KO3P131(c)






P131

AP

IH

36 h

9 h

ΔMoKMT2H

(a)

0

20

40

60

80

100

120

P131 ΔMoKMT2H

Con

idiu

m fo

rmat

ion

rate

(b)

P131



(c)



(d)










E

E

E

E

Eg

G

G

G

G

Gg

G

G

G

G

Gr

R

R

R

R

Ri

I

I

I

I

Ie

E

E

E

E

Ey

Y

Y

Y

Y

Yg

G

G

G

G

Ge

E

E

E

E

Ee

E

E

E

E

Ee

E

E

E

E

E






r

R

R

R

R

R

m

M

M

M

M

M

y

Y

Y

Y

Y

Y

y

Y

Y

Y

Y

Y

d

D

D

D

D

D

d

D

D

D

D

D

g

G

G

G

G

G

r

R

R

R

R

R

f

F

F

F

F

F

n

N

N

N

N

N

h

H

H

H

H

H

s

S

S

S

S

S

c

C

C

C

C

C






p

P

P

P

P

P

n

N

N

N

N

N

c

C

C

C

C

C

m

M

M

M

M

M

k

K

K

K

K

K

w

W

W

W

W

W

v

V

V

V

V

V

g

G

G

G

G

G

r

R

R

R

R

R

l

L

L

L

L

L

a

A

A

A

A

A

g

G

G

G

G

G

e

E

E

E

E

E

l

L

L

L

L

L

t

T

T

T

T

T

y

Y

Y

Y

Y

Y

d

D

D

D

D

D

y

Y

Y

Y

Y

Y

n

N

N

N

N

N

f

F

F

F

F

F

f

F

F

F

F

F






q

Q

Q

Q

Q

Q

c

C

C

C

C

C

c

C

C

C

C

C

c

C

C

C

C

C

r

R

R

R

R

R

g

G

G

G

G

G

g

G

G

G

G

G


SET domain

SET domain




40

40

40

40

40

80

80

80

79

79

119

119

119

119

119

140

140

140

140

140

Zn2+ binding motif





4 Discussion



H3K4me3

H3K9me3

H3K27me3

H3K36me3

H3K36me2

H3

P131 ΔMoKMT2H




Competing Interests




Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








3 Results



HYG-LBCK

ATG TAA

LBCK

LB-R

HYG

HYG-R1

Probe

EcoRI EcoRI

Genomic DNA

HYG-R

LB-F

HYG-F

RB-R RBCK

HYG-RRB-F

HY

YG

HYG-F1

LB

RB

LB

LBCK

RBCKUPRUPF

HYG RB

HYG-RBCK

MoKMT2H

400 bp

(a)

39 Kb

65 Kb

231 Kb

94 Kb

65 Kb

43 Kb

23 Kb20 Kb

KO3KO2KO1P131Marker

(b)

11 Kb

26 KbUPF+HYG-R

UPR+HYG-F

P131 KO2 KO3

(c)





P131 KO2 KO3

(a)

50

100

150

200

250

300

350

400

450

Col

ony

diam

eter

(mm

)

KO2 KO3P131(b)

10

20

30

40

50

60

Num

ber o

f con

idia

(107)

KO2 KO3P131(c)






P131

AP

IH

36 h

9 h

ΔMoKMT2H

(a)

0

20

40

60

80

100

120

P131 ΔMoKMT2H

Con

idiu

m fo

rmat

ion

rate

(b)

P131



(c)



(d)










E

E

E

E

Eg

G

G

G

G

Gg

G

G

G

G

Gr

R

R

R

R

Ri

I

I

I

I

Ie

E

E

E

E

Ey

Y

Y

Y

Y

Yg

G

G

G

G

Ge

E

E

E

E

Ee

E

E

E

E

Ee

E

E

E

E

E






r

R

R

R

R

R

m

M

M

M

M

M

y

Y

Y

Y

Y

Y

y

Y

Y

Y

Y

Y

d

D

D

D

D

D

d

D

D

D

D

D

g

G

G

G

G

G

r

R

R

R

R

R

f

F

F

F

F

F

n

N

N

N

N

N

h

H

H

H

H

H

s

S

S

S

S

S

c

C

C

C

C

C






p

P

P

P

P

P

n

N

N

N

N

N

c

C

C

C

C

C

m

M

M

M

M

M

k

K

K

K

K

K

w

W

W

W

W

W

v

V

V

V

V

V

g

G

G

G

G

G

r

R

R

R

R

R

l

L

L

L

L

L

a

A

A

A

A

A

g

G

G

G

G

G

e

E

E

E

E

E

l

L

L

L

L

L

t

T

T

T

T

T

y

Y

Y

Y

Y

Y

d

D

D

D

D

D

y

Y

Y

Y

Y

Y

n

N

N

N

N

N

f

F

F

F

F

F

f

F

F

F

F

F






q

Q

Q

Q

Q

Q

c

C

C

C

C

C

c

C

C

C

C

C

c

C

C

C

C

C

r

R

R

R

R

R

g

G

G

G

G

G

g

G

G

G

G

G


SET domain

SET domain




40

40

40

40

40

80

80

80

79

79

119

119

119

119

119

140

140

140

140

140

Zn2+ binding motif





4 Discussion



H3K4me3

H3K9me3

H3K27me3

H3K36me3

H3K36me2

H3

P131 ΔMoKMT2H




Competing Interests




Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


HYG-LBCK

ATG TAA

LBCK

LB-R

HYG

HYG-R1

Probe

EcoRI EcoRI

Genomic DNA

HYG-R

LB-F

HYG-F

RB-R RBCK

HYG-RRB-F

HY

YG

HYG-F1

LB

RB

LB

LBCK

RBCKUPRUPF

HYG RB

HYG-RBCK

MoKMT2H

400 bp

(a)

39 Kb

65 Kb

231 Kb

94 Kb

65 Kb

43 Kb

23 Kb20 Kb

KO3KO2KO1P131Marker

(b)

11 Kb

26 KbUPF+HYG-R

UPR+HYG-F

P131 KO2 KO3

(c)





P131 KO2 KO3

(a)

50

100

150

200

250

300

350

400

450

Col

ony

diam

eter

(mm

)

KO2 KO3P131(b)

10

20

30

40

50

60

Num

ber o

f con

idia

(107)

KO2 KO3P131(c)






P131

AP

IH

36 h

9 h

ΔMoKMT2H

(a)

0

20

40

60

80

100

120

P131 ΔMoKMT2H

Con

idiu

m fo

rmat

ion

rate

(b)

P131



(c)



(d)










E

E

E

E

Eg

G

G

G

G

Gg

G

G

G

G

Gr

R

R

R

R

Ri

I

I

I

I

Ie

E

E

E

E

Ey

Y

Y

Y

Y

Yg

G

G

G

G

Ge

E

E

E

E

Ee

E

E

E

E

Ee

E

E

E

E

E






r

R

R

R

R

R

m

M

M

M

M

M

y

Y

Y

Y

Y

Y

y

Y

Y

Y

Y

Y

d

D

D

D

D

D

d

D

D

D

D

D

g

G

G

G

G

G

r

R

R

R

R

R

f

F

F

F

F

F

n

N

N

N

N

N

h

H

H

H

H

H

s

S

S

S

S

S

c

C

C

C

C

C






p

P

P

P

P

P

n

N

N

N

N

N

c

C

C

C

C

C

m

M

M

M

M

M

k

K

K

K

K

K

w

W

W

W

W

W

v

V

V

V

V

V

g

G

G

G

G

G

r

R

R

R

R

R

l

L

L

L

L

L

a

A

A

A

A

A

g

G

G

G

G

G

e

E

E

E

E

E

l

L

L

L

L

L

t

T

T

T

T

T

y

Y

Y

Y

Y

Y

d

D

D

D

D

D

y

Y

Y

Y

Y

Y

n

N

N

N

N

N

f

F

F

F

F

F

f

F

F

F

F

F






q

Q

Q

Q

Q

Q

c

C

C

C

C

C

c

C

C

C

C

C

c

C

C

C

C

C

r

R

R

R

R

R

g

G

G

G

G

G

g

G

G

G

G

G


SET domain

SET domain




40

40

40

40

40

80

80

80

79

79

119

119

119

119

119

140

140

140

140

140

Zn2+ binding motif





4 Discussion



H3K4me3

H3K9me3

H3K27me3

H3K36me3

H3K36me2

H3

P131 ΔMoKMT2H




Competing Interests




Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


P131 KO2 KO3

(a)

50

100

150

200

250

300

350

400

450

Col

ony

diam

eter

(mm

)

KO2 KO3P131(b)

10

20

30

40

50

60

Num

ber o

f con

idia

(107)

KO2 KO3P131(c)






P131

AP

IH

36 h

9 h

ΔMoKMT2H

(a)

0

20

40

60

80

100

120

P131 ΔMoKMT2H

Con

idiu

m fo

rmat

ion

rate

(b)

P131



(c)



(d)










E

E

E

E

Eg

G

G

G

G

Gg

G

G

G

G

Gr

R

R

R

R

Ri

I

I

I

I

Ie

E

E

E

E

Ey

Y

Y

Y

Y

Yg

G

G

G

G

Ge

E

E

E

E

Ee

E

E

E

E

Ee

E

E

E

E

E






r

R

R

R

R

R

m

M

M

M

M

M

y

Y

Y

Y

Y

Y

y

Y

Y

Y

Y

Y

d

D

D

D

D

D

d

D

D

D

D

D

g

G

G

G

G

G

r

R

R

R

R

R

f

F

F

F

F

F

n

N

N

N

N

N

h

H

H

H

H

H

s

S

S

S

S

S

c

C

C

C

C

C






p

P

P

P

P

P

n

N

N

N

N

N

c

C

C

C

C

C

m

M

M

M

M

M

k

K

K

K

K

K

w

W

W

W

W

W

v

V

V

V

V

V

g

G

G

G

G

G

r

R

R

R

R

R

l

L

L

L

L

L

a

A

A

A

A

A

g

G

G

G

G

G

e

E

E

E

E

E

l

L

L

L

L

L

t

T

T

T

T

T

y

Y

Y

Y

Y

Y

d

D

D

D

D

D

y

Y

Y

Y

Y

Y

n

N

N

N

N

N

f

F

F

F

F

F

f

F

F

F

F

F






q

Q

Q

Q

Q

Q

c

C

C

C

C

C

c

C

C

C

C

C

c

C

C

C

C

C

r

R

R

R

R

R

g

G

G

G

G

G

g

G

G

G

G

G


SET domain

SET domain




40

40

40

40

40

80

80

80

79

79

119

119

119

119

119

140

140

140

140

140

Zn2+ binding motif





4 Discussion



H3K4me3

H3K9me3

H3K27me3

H3K36me3

H3K36me2

H3

P131 ΔMoKMT2H




Competing Interests




Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


P131

AP

IH

36 h

9 h

ΔMoKMT2H

(a)

0

20

40

60

80

100

120

P131 ΔMoKMT2H

Con

idiu

m fo

rmat

ion

rate

(b)

P131



(c)



(d)










E

E

E

E

Eg

G

G

G

G

Gg

G

G

G

G

Gr

R

R

R

R

Ri

I

I

I

I

Ie

E

E

E

E

Ey

Y

Y

Y

Y

Yg

G

G

G

G

Ge

E

E

E

E

Ee

E

E

E

E

Ee

E

E

E

E

E






r

R

R

R

R

R

m

M

M

M

M

M

y

Y

Y

Y

Y

Y

y

Y

Y

Y

Y

Y

d

D

D

D

D

D

d

D

D

D

D

D

g

G

G

G

G

G

r

R

R

R

R

R

f

F

F

F

F

F

n

N

N

N

N

N

h

H

H

H

H

H

s

S

S

S

S

S

c

C

C

C

C

C






p

P

P

P

P

P

n

N

N

N

N

N

c

C

C

C

C

C

m

M

M

M

M

M

k

K

K

K

K

K

w

W

W

W

W

W

v

V

V

V

V

V

g

G

G

G

G

G

r

R

R

R

R

R

l

L

L

L

L

L

a

A

A

A

A

A

g

G

G

G

G

G

e

E

E

E

E

E

l

L

L

L

L

L

t

T

T

T

T

T

y

Y

Y

Y

Y

Y

d

D

D

D

D

D

y

Y

Y

Y

Y

Y

n

N

N

N

N

N

f

F

F

F

F

F

f

F

F

F

F

F






q

Q

Q

Q

Q

Q

c

C

C

C

C

C

c

C

C

C

C

C

c

C

C

C

C

C

r

R

R

R

R

R

g

G

G

G

G

G

g

G

G

G

G

G


SET domain

SET domain




40

40

40

40

40

80

80

80

79

79

119

119

119

119

119

140

140

140

140

140

Zn2+ binding motif





4 Discussion



H3K4me3

H3K9me3

H3K27me3

H3K36me3

H3K36me2

H3

P131 ΔMoKMT2H




Competing Interests




Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







E

E

E

E

Eg

G

G

G

G

Gg

G

G

G

G

Gr

R

R

R

R

Ri

I

I

I

I

Ie

E

E

E

E

Ey

Y

Y

Y

Y

Yg

G

G

G

G

Ge

E

E

E

E

Ee

E

E

E

E

Ee

E

E

E

E

E






r

R

R

R

R

R

m

M

M

M

M

M

y

Y

Y

Y

Y

Y

y

Y

Y

Y

Y

Y

d

D

D

D

D

D

d

D

D

D

D

D

g

G

G

G

G

G

r

R

R

R

R

R

f

F

F

F

F

F

n

N

N

N

N

N

h

H

H

H

H

H

s

S

S

S

S

S

c

C

C

C

C

C






p

P

P

P

P

P

n

N

N

N

N

N

c

C

C

C

C

C

m

M

M

M

M

M

k

K

K

K

K

K

w

W

W

W

W

W

v

V

V

V

V

V

g

G

G

G

G

G

r

R

R

R

R

R

l

L

L

L

L

L

a

A

A

A

A

A

g

G

G

G

G

G

e

E

E

E

E

E

l

L

L

L

L

L

t

T

T

T

T

T

y

Y

Y

Y

Y

Y

d

D

D

D

D

D

y

Y

Y

Y

Y

Y

n

N

N

N

N

N

f

F

F

F

F

F

f

F

F

F

F

F






q

Q

Q

Q

Q

Q

c

C

C

C

C

C

c

C

C

C

C

C

c

C

C

C

C

C

r

R

R

R

R

R

g

G

G

G

G

G

g

G

G

G

G

G


SET domain

SET domain




40

40

40

40

40

80

80

80

79

79

119

119

119

119

119

140

140

140

140

140

Zn2+ binding motif





4 Discussion



H3K4me3

H3K9me3

H3K27me3

H3K36me3

H3K36me2

H3

P131 ΔMoKMT2H




Competing Interests




Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


H3K4me3

H3K9me3

H3K27me3

H3K36me3

H3K36me2

H3

P131 ΔMoKMT2H




Competing Interests




Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article An Ash1-Like Protein MoKMT2H Null Mutant Is …downloads.hindawi.com/journals/bmri/2016/1575430.pdf · 2019-07-30 · Research Article An Ash1-Like Protein MoKMT2H