DEMETER Plays A Role In DNA Demethylation And Disease .

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EpigeneticsISSN: 1559-2294 (Print) 1559-2308 (Online) Journal homepage: https://www.tandfonline.com/loi/kepi20DEMETER plays a role in DNA demethylation anddisease response in somatic tissues of ArabidopsisUlrike Schumann, Joanne M. Lee, Neil A. Smith, Chengcheng Zhong, JianKang Zhu, Elizabeth S. Dennis, Anthony A. Millar & Ming-Bo WangTo cite this article: Ulrike Schumann, Joanne M. Lee, Neil A. Smith, Chengcheng Zhong, JianKang Zhu, Elizabeth S. Dennis, Anthony A. Millar & Ming-Bo Wang (2019): DEMETER plays a rolein DNA demethylation and disease response in somatic tissues of Arabidopsis, Epigenetics, DOI:10.1080/15592294.2019.1631113To link to this article: https://doi.org/10.1080/15592294.2019.1631113View supplementary materialAccepted author version posted online: 12Jun 2019.Published online: 19 Jun 2019.Submit your article to this journalArticle views: 108View Crossmark dataFull Terms & Conditions of access and use can be found ation?journalCode kepi20

631113RESEARCH PAPERDEMETER plays a role in DNA demethylation and disease response in somatictissues of ArabidopsisUlrike Schumann a*†, Joanne M. Leea*‡, Neil A. Smitha, Chengcheng Zhonga, Jian-Kang Zhub,Elizabeth S. Dennisa, Anthony A. Millarc, and Ming-Bo WangaaCSIRO Agriculture and Food, Acton, Australia; bDepartment of Horticulture and Landscape Architecture, Purdue University, West Lafayette,IN, USA; cResearch School of Biology, Australian National University, Acton, AustraliaABSTRACTARTICLE HISTORYDNA demethylases function in conjunction with DNA methyltransferases to modulate genomic DNAmethylation levels in plants. The Arabidopsis genome contains four DNA demethylase genes, DEMETER(DME), REPRESSOR OF SILENCING 1 (ROS1) also known as DEMETER-LIKE 1 (DML1), DML2, and DML3. WhileROS1, DML2, and DML3 were shown to function in disease response in somatic tissues, DME has beenthought to function only in reproductive tissues to maintain the maternal-specific expression pattern ofa subset of imprinted genes. Here we used promoter:β-glucuronidase (GUS) fusion constructs to showthat DME is constitutively expressed throughout the plant, and that ROS1, DML2, and DML3 have tissuespecific expression patterns. Loss-of-function mutations in DME cause seed abortion and thereforeviable DME mutants are not available for gene function analysis. We knocked down DME expression ina triple ros1 dml2 dml3 (rdd) mutant background using green tissue-specific expression of a hairpin RNAtransgene (RNAi), generating a viable ‘quadruple’ demethylase mutant line. We show that this rdd DMERNAi line has enhanced disease susceptibility to Fusarium oxysporum infection compared to the rddtriple mutant. Furthermore, several defence-related genes, previously shown to be repressed in rdd,were further repressed in the rdd DME RNAi plants. DNA methylation analysis of two of these genesrevealed increased differential promoter DNA methylation in rdd DME RNAi plants compared to WT,beyond the difference observed in the parental rdd plants. These results indicate that DME contributesto DNA demethylase activity and disease response in somatic tissues.Received 4 March 2019Revised 28 May 2019Accepted 5 June 2019IntroductionDNA cytosine methylation is a major epigeneticmark in eukaryotes. In plants, the DNA methylation level in the genome is controlled by de novoDNA methylation, maintenance DNA methylationand DNA demethylation. De novo methylation ismediated by RNA-directed DNA methylation(RdDM), which can occur at all cytosine contexts,namely CG, CHG (‘H’ stands for A, C, T) andCHH sites. Methylation at symmetric CG andCHG sites can be maintained during DNA replication by METHYLTRANSFERASE 1 (MET1) andCHROMOMETHYLASE 3 (CMT3), respectively.CHH methylation at highly repetitive regions ofthe genome can be maintained by CMT2, butCHH methylation near genes cannot be maintained during DNA replication and depends onKEYWORDSDEMETER; DEMETER-LIKE;ROS1; rdd; DNAdemethylation; Fusarium;Arabidopsis thaliana;Epigeneticscontinuous RdDM. DNA demethylation canoccur passively during DNA replication whenmaintenance methylation is incomplete, orthrough the activity of the DNA glycosylase familyof DNA demethylases. These DNA demethylasesremove 5-methylcytosine and replace it withunmethylated cytosine through a base-excisionrepair mechanism [1].The Arabidopsis genome contains four DNAdemethylase genes, DEMETER (DME), REPRESSOROF SILENCING 1 (ROS1) also known as DEMETERLIKE 1 (DML1), DML2, and DML3 [1]. DME hasbeen shown to play a major role in seed developmentby controlling the expression of specific imprintedmaternal alleles, and mutation of the maternal copyof the DME gene leads to seed abortion [2,3]. ROS1,DML2, and DML3 are thought to account for allCONTACT Ming-Bo Wangming-bo.wang@csiro.auCSIRO Agriculture and Food, Clunies Ross Street, Acton, ACT 2601, Australia*These authors contributed equally to this work†Present address: EMBL-Australia Collaborating Group, John Curtin School of Medical Research, Australian National University, Acton, ACT 2601, Australia‡Present address: Plant Health Australia, Deakin, ACT 2600, AustraliaSupplemental data for this article can be accessed here. 2019 Informa UK Limited, trading as Taylor & Francis Group

2U. SCHUMANN ET AL.demethylase activities in somatic tissues. ROS1 is thedominant demethylase of the three and plays a role inmaintaining the activity of transgenes and transposable elements in Arabidopsis by decreasing DNAmethylation [1]. Both localized and genome-wideDNA methylation analyses have shown that DML2and DML3 function redundantly with ROS1 to maintain low-level cytosine methylation at some loci [4,5].DML2 and DML3 have also been shown to play a rolein modulating the DNA methylation level of someheavily methylated loci in Arabidopsis [6]. In additionto maintaining genome methylation levels, the threeDNA demethylase genes have recently been implicated in plant disease resistance in Arabidopsis. Thesingle mutant ros1, and the triple mutant ros1 dml2dml3 (rdd), have been shown to display increasedsusceptibility to the bacterial pathogen Pseudomonassyringae [7] and the fungal pathogen Fusarium oxysporum [8], suggesting that these DNA demethylasesplay a role in plant defence responses by regulatingplant defence-related genes.The expression patterns of the four DNA demethylase genes have been investigated. Choi et al. [3]reported that DME is expressed specifically in thecentral cells of female gametophytes, but is undetectable in leaf tissues using RT-PCR. In contrast, OrtegaGallisteo et al. [6] detected the constitutive expressionof DME as well as ROS1, DML2 and DML3 acrossvarious plant tissues using RT-PCR. Further, theyused promoter:β-glucuronidase (GUS) constructs toexamine the expression pattern of DML2 and DML3,and showed that both genes are strongly expressed inall tissues of Arabidopsis. Gong et al. [9] also usedpromoter:GUS reporter constructs to investigate theexpression pattern of ROS1, and showed strongexpression in all tissues of young Arabidopsis seedlings. DME, ROS1 and DML2 expression patterns arealso represented in the Arabidopsis Gene ExpressionVisualization database (AtGenExpress [10]), whichshows constitutive expression of DME but more variable expression levels of ROS1 and DML2 across different plant tissues (Supplementary Figure S1A). NoDML3 expression data were recorded as DML3 wasnot represented on the Affymetrix ATH-1 array.However, DML3 RNA sequencing data are availablein the Transcriptome Variation Analysis database(TraVA [11]), which shows the localized expressionof DML3 in anthers only. In support of this, ourprevious microarray expression analysis [8] andmRNA sequencing (unpublished) of 3-week-oldwhole Col-0 Arabidopsis seedlings detected the lowlevel expression of DML3 along with the relativelyhigh-level expression of DME, ROS1 and DML2(Supplementary Figure S1B, S1C). This contradictsthe reported constitutive expression pattern ofDML3 [6]. Thus, the expression patterns of the fourdemethylase genes require further examination, but itseems clear that DME has a widespread expression inboth reproductive and vegetative tissues ofArabidopsis.The observed DME expression pattern raised thepossibility that DME not only functions in reproductive tissue but also contributes to DNA demethylaseactivity and related biological function in the wholeplant. However, this has not been investigated, possibly because a loss-of-function mutation in DMEresults in seed abortion, and therefore double, triple,or quadruple demethylase mutants containing a DMEmutation are not available for genomic and biologicalfunction studies. Indeed, genome-wide methylationand gene expression studies utilized either singleDNA demethylase mutants or the triple demethylasemutant rdd. Both DNA methylation and gene expression changes in rdd are limited, with a relatively smallnumber of loci affected by the mutations [4,12,13].The functional analysis of the DNA demethylases indisease resistance has also been based on the use of thesingle ros1 and triple rdd mutants [5,7,8].The present study was aimed at examining thebiological function of DME in somatic tissues. Usingpromoter:GUS fusion constructs, we confirmed theconstitutive expression pattern of DME and revealedtissue-differential or tissue-specific expression patterns of ROS1, DML2 and DML3. We knockeddown the expression of DME in the rdd mutant background using green tissue-specific RNAi to generatea viable rdd DME RNAi line. We used this line todemonstrate that DME plays a role in Fusarium resistance, defence gene expression and DNA demethylation in vegetative Arabidopsis tissues.ResultsDME, ROS1, DML2 and DML3 promoters showdistinct expression patternsThe structures of the promoter:GUS fusion constructs are illustrated in Figure 1. The promoter

EPIGENETICSRBNos-TDME NPTII:Nos-TRBNos-P:NPTII:Nos-TDML2 promotersDMESSU promoterNos-TLBNos-TDML3 promoter-3858-1555LB-1-2891RBNos-T 535-2777RBLB 584ROS1 promoterNos-P:NPTII:Nos-TLB 584 TE DME promoterNos-P:NPTII:Nos-T3LB-43PDK intronasDMEOCS-TNos-P:BAR:Nos-TLB 171Figure 1. Schematic diagrams of the promoter:GUS fusion constructs and the DME hairpin RNA (hpDME) construct. Transposableelement (TE) sequences are indicated in brown; blue regions indicate the GUS coding sequence. Numbers indicate the nucleotideposition relative to the transcription start site (TSS).fragments were amplified from the genomic regionupstream of the translation start site ATG, withthe size of 2.90 and 3.56 kb (DME; 2321 to 584and 2972 to 584 relative to the transcriptionstart site or TSS), 3.31 kb (ROS1; 2777 to 535),2.89 kb (DML2; 2891 to 1), and 3.82 kb (DML3; 3858 to 43), respectively. These promoter fragments contain longer upstream sequences than thepreviously used ones for DME ( 2282 to 1922 bpof the first exon) [3], ROS1 ( 1565 to 25 fromfirst ATG or 1030 to 510 relative to TSS) [9],DML2 ( 2269 to 15 from first ATG or TSS) andDML3 ( 1300 to 13 from first ATG or 1145 to 171 relative to TSS) [6] promoters, respectively.Two versions of the DME promoter were tested;one included the full-length upstream transposableelement (TE) sequence (DME TEp) in case the TEplays a role in DME regulation, and the second(DMEp) contained part of the TE but excludingthe upstream section that overlaps with the transcription start site of the adjacent gene. The ROS1,DML2 and DML3 promoter fragments also contained TE sequence that exists in the upstreamgenomic sequences of the respective genes(Figure 1). To avoid the potential enhancer effectsby strong constitutive promoters such as the 35Spromoter [14], the promoter:GUS expression cassettes were cloned into the plant expression vectorpBI101, where the selective marker gene NPTII isdriven by the relatively weak nopaline synthasepromoter (Nos-P), which is located distal to theinserted endogenous gene promoters (Figure 1).Transgenic lines of the Col-0 ecotype wereobtained, self-fertilized until the fourth generation,and then analysed for GUS expression patterns. Asshown in Figure 2 and Supplementary Figure S2, theDME promoter lines showed strong GUS staining inall tissues analysed, including leaves, roots, flowersand siliques. No visible difference in GUS stainingcould be observed between the two versions of theDME promoter constructs, DME TEp:GUS andDMEp:GUS. In addition, quantitative expressionanalysis of four independent lines for each of theDME constructs, using a fluorometric MUG assay,detected no significant difference in GUS expressionlevels between the two (Supplementary Figure S3).This indicated that the additional upstream TEsequence in DME TEp:GUS is not required forDME expression. The GUS expression pattern ofthe two DME promoter constructs confirmed thatDME is transcribed throughout the reproductive andvegetative tissues of Arabidopsis, which is consistentwith the RT-PCR result of Ortega-Gallisteo et al. [6].The ROS1p:GUS promoter construct showeda much lower expression than the DMEp:GUSpromoter constructs and displayed distinct expression patterns in different tissues. The expressionlevel was relatively high in young leaves, particularly in the midrib areas, and in young flowers andsiliques, but weaker in roots and older leaves andsiliques (Figure 2 and Supplementary Figure S2).This was consistent with the ROS1 expression pattern recorded in the AtGenExpress and TraVadatabases [10,11], which showed relatively high

4U. SCHUMANN ET AL.DME TEp:GUS (L#2)DMEp:GUS (L#2)ROS1p:GUS (L#4)DML2p:GUS (L#2)DML3p:GUS (L#2)Figure 2. Representative expression patterns of the promoter:GUS fusion transgenes in Arabidopsis Col-0 ecotype. Arrow indicatesDML3p:GUS expression in anthers of young flowers.

EPIGENETICSlevels in the shoot apex and flower tissues but lowlevels in root, stem and leaves (SupplementaryFigure S1A).The DML2p:GUS construct showed weakexpression in leaves and roots, with clear GUSstaining mainly in the leaf midrib areas.However, it was strongly expressed in the flowersand siliques (Figure 2 and Supplementary FigureS2). This result indicated that DML2 is predominantly expressed in reproductive tissues.The DML3p:GUS construct displayed the mosttissue-specific expression pattern among thedemethylase gene promoter constructs. The DML3p:GUS lines showed no detectable GUS staining inyoung seedlings, siliques or most flower tissues(Figure 2 and Supplementary Figure S2). However,clear GUS staining was consistently detected in young(but not old) anther tissues after an extended periodof staining (up to three days), which matched the datarecorded in the TraVa database [11]. Thus, DML3 islikely to be an anther-specific demethylase gene.RNA interference knockdown of DME in rddincreases plant susceptibility to Fusariumoxysporum infectionIn order to examine if DME contributes to DNAdemethylase function in somatic tissues, a hairpinRNA construct (hpDME) was designed to silenceDME specifically in green tissues. We used theArabidopsis rubisco small subunit gene promoter(SSU) to drive the expression of hpDME (Figure 1).The 548 bp sequence of the DME cDNA in thehpDME construct had two short (20 and 21 nt)stretches of sequence identity with DML2 butshowed no high nucleotide sequence similarities toROS1 and DML3. This construct was transformedinto rdd plants to achieve DME knockdown in thetriple demethylase mutant background. We alsointroduced the construct into Col-0 to obtain DMEknockdown lines in the wild-type background.Transgenic lines containing the hpDME constructshowed no phenotypic difference to the untransformed rdd or the wild-type Col-0 plants, and werefully fertile (data not shown). Two single-copy transgenic lines in the rdd background, rdd-HP5 and rddHP6, were self-fertilized to generate homozygousprogeny populations and used for disease response,gene expression and DNA methylation analyses.5Two hpDME lines of the Col-0 background, ColHP4 and Col-HP7, were also included. Northernblot analysis of the target DME mRNA showed thatrdd-HP5 plants had little hpDME expression, henceonly a slight reduction in DME mRNA level wasobserved (Figure 3 and Supplementary Figure S4).rdd-HP6 plants, on the other hand, showed a highlevel of hpDME expression and concomitant strongdownregulation of DME mRNA. Similarly, Col-HP4showed clear hpDME expression and DME downregulation, but Col-HP7 had little hpDME expression or DME downregulation (SupplementaryFigure S4). It was notable that lower-molecularweight DME-specific hybridizing signals were alsodetected on the northern blots, but the nature ofthese bands remained unclear.Our previous study showed that rdd plants haveincreased susceptibility to the fungal pathogenFusarium oxysporum compared to Col-0 [5,8],suggesting that DNA demethylases play a positiverole in Fusarium resistance. To examine if DMEcontributes to disease resistance, the homozygousT3 and T4 generations of rdd-HP5 and rdd-HP6transgenic lines were assayed for disease susceptibility to Fusarium, along with Col-0 and rddplants as controls. As shown in Figure 4, the rddHP6 plants, with strong downregulation of DME,showed a significant increase in the chlorotic disease phenotype compared with the parental rddline, whereas rdd-HP5 plants, with only slightdownregulation of DME, displayed a diseaseresponse similar to rdd. Soil infections yieldedcomparable results for these lines (SupplementaryFigure S5). The Col-HP4 or Col-HP7 lines showedno detectable increase in Fusarium disease phenotypes compared to the Col-0 plant (SupplementaryFigure S6), suggesting that knockdown of DMEexpression alone is insufficient to affect plantresponse to Fusarium infection.DME contributes to gene expression regulationand DNA demethylation of promoter TEs ofdefence-related genesOur previous study has identified a subset ofplant defence-related genes that are regulated byDNA demethylases in Arabidopsis: they containtransposable element (TE) sequences in their promoters and show differential DNA methylation in

6U. SCHUMANN ET )(TDME mRNAhpRNArRNAFigure 3. Northern blot analysis to detect DME mRNA level in Col-0, rdd and rdd DME RNAi transformants (rdd-HP5 and rdd-HP6). Toppanel: Northern blot analysis probed with antisense DME RNA, which allows detection of both the DME mRNA and the hpDMEderived transcript. Bottom panel: Ethidium bromide-stained RNA agarose gel to show even loading of RNA.the promoter TEs and repressed gene expressionin rdd compared to Col-0 [5,8]. Among the threeDNA demethylase genes, ROS1 plays a dominantrole, but DML2 and DML3 also function redundantly to regulate these genes [5]. To investigateif DME also contributes to defence gene regulation, we selected a number of these rdddownregulated defence-related genes and compared their expression in Col-0, rdd, andhpDME lines in the rdd and Col-0 backgrounds.As shown in Figure 5, these defence-relatedgenes were downregulated in the rdd mutant compared to Col-0 plants, consistent with our previousfindings [5,8]. RNAi-mediated knockdown ofDME in Col-0 did not affect the expression ofthese genes (except for AT1G05700 whichappeared to show slight downregulation in theCol-0 DME RNAi lines), which was consistentwith the Fusarium infection result (Figure S6).This suggested that downregulation of DMEalone is insufficient, or the level of DME downregulation in this line is not high enough, to affectthe expression of these genes or the diseaseresponses. However, RNAi-mediated downregulation of DME in the rdd background clearlyaffected the expression of all six defence-relatedgenes analysed, which showed increased repression compared to the parental rdd plants (Figure5). Consistent with the greater DME knockdownin line rdd-HP6 compared to rdd-HP5, all analysed defence-related genes showed strongerdownregulation in the rdd-

DNA cytosine methylation is a major epigenetic mark in eukaryotes. In plants, the DNA methyla-tion level in the genome is controlled by de novo DNA methylation, maintenance DNA methylation and DNA demethylation. De novo methylation is mediated by RNA-directed DNA methylation (RdDM), which can occur at all cytosine contexts,

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