Diversity, Evolution and Expression Profiles of Histone Acetyltransferases and Deacetylases in Oomycetes Xiao-Wen Wang, Li-Yun Guo*, Miao Han and Kun Shan

Wang et al. BMC Genomics (2016) 17:927 DOI 10.1186/s12864-016-3285-y

RESEARCHARTICLE Open Access Diversity, evolution and expression profiles of histone acetyltransferases and deacetylases in oomycetes Xiao-Wen Wang, Li-Yun Guo*, Miao Han and Kun Shan

Abstract Background: Oomycetes are a group of fungus-like eukaryotes with diverse microorganisms living in marine, freshwater and terrestrial environments. Many of them are important pathogens of plants and animals, causing severe economic losses. Based on previous study, gene expression in eukaryotic cells is regulated by epigenetic mechanisms such as DNA methylation and histone modification. However, little is known about epigenetic mechanisms of oomycetes. Results: In this study, we investigated the candidate genes in regulating histone acetylation in oomycetes genomes through bioinformatics approaches and identified a group of diverse histone acetyltransferases (HATs) and histone deacetylases (HDACs), along with three putative novel HATs. Phylogenetic analyses suggested that most of these oomycetes HATs and HDACs derived from distinct evolutionary ancestors. Phylogenetic based analysis revealed the complex and distinct patterns of duplications and losses of HATs and HDACs in oomycetes. Moreover, gene expression analysis unveiled the specific expression patterns of the 33 HATs and 11 HDACs of Phytophthora infestans during the stages of development, infection and stress response. Conclusions: In this study, we reveal the structure, diversity and the phylogeny of HATs and HDACs of oomycetes. By analyzing the expression data, we provide an overview of the specific biological stages of these genes involved. Our datasets provide useful inputs to help explore the epigenetic mechanisms and the relationship between genomes and phenotypes of oomycetes. Keywords: Oomycetes, Histone acetyltransferase, Histone deacetylase, Epigenetics, Living habitats, Expression profiles

Background more 5-methylcytosines have lower transcriptional activity In eukaryotes, gene expression and physiological function [7]. However, no evidence of the regulation of gene can be regulated by epigenetic mechanisms. Epigenetic expression associated with DNA methylation in oomy- modifications of chromatin can cause heritable changes cetes is documented [8, 9]. that are not encoded by the underlying DNA sequences. Histone proteins are the core particles of nucleosomes The mechanisms involved mainly include DNA methyla- that are packed together to form eukaryotic chromatins tion and histone modification [1]. [10]. The N-terminal tails of histones can extend from the DNA methylation mostly occurs at CpG dinucleotides nucleosomes and can be modified post-translationally through adding methyl groups and converting cytosine to (PTMs) by mechanisms such as acetylation, methylation, 5-methylcytosine by DNA methyltransferases (DNMTs) phosphorylation, ubiquitination, and sumoylation by dif- [2, 3]. DNMT1, DNMT2, DNMT3A, DNMT3B, and ferent histone modifiers [11]. Among the PTMs, histone DNMT3L are C(5)-cytosine-specific DNA methyltransfer- acetylation at lysine residues is well characterized in yeast, ases [4–6]. A research has shown that promoters with plants and animals [12, 13]. Histone acetylation occurs at the amino groups of the lysine residues on the N- * Correspondence: [email protected]; [email protected] terminus of histone tails. The dynamic PTM histone lysine Department of Plant Pathology and the Ministry of Agriculture Key Laboratory for Plant Pathology, China Agricultural University, Beijing 100193, acetylation is regulated by histone acetyltransferases China (HATs) and histone deacetylases (HDACs) [13–15]. HATs

© The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wang et al. BMC Genomics (2016) 17:927 Page 2 of 14

transfer an acetyl moiety from acetyl coenzyme A (acetyl and animals, causing severe economic losses. Examples CoA) to the Ɛ-amino group of specific lysine residues on include Phytophthora infestans, the causal pathogen of histone N-terminal tails [16, 17]. As a consequence, potato late blight, which resulted in the death of millions histone acetylation promotes RNA polymerase and other of people in the Irish potato famine in the 19th century; transcriptional factor complexes to interact with DNA the sudden oak death pathogen Phytophthora ramorum [18], and leads to increased gene expression [19]. In and Jarrah forest dieback pathogen Phytophthora cin- contrast, HDACs remove acetyl modifications from his- namomi, which affect a large variety of woody plants tones and lead to down-regulated gene expression. Gene resulting in natural and ecosystems damage [35, 36]; and families of both HATs and HDACs are evolutionarily Saprolegnia parasitica, a devastating pathogen of many conserved in eukaryotes [20–22] and have been shown to freshwater fish [37]. Although most oomycetes have play a crucial role in transcriptional regulation in yeast, nutritional and ecological characteristics similar to the plants and mammals [11]. true fungi, several biochemical and cytological features Based on intracellular localization and substrate speci- distinguish them from the true fungi [38]. For instance, ficity, HATs are classified into two categories, type A (i) their cell walls are composed of cellulose and glycan and type B [23]. The type A HATs are usually localized instead of chitin; (ii) their mitochondria contain tubular in the nuclei and are involved in transcriptional regula- cristae instead of disc-like cristae; (iii) their nuclei are tion by modification of specific lysine residues on his- diploid in asexual stage; and (iv) they are sterol auxo- tone tails [24]. According to sequence conservation in trophs. Concrete evidence from molecular phylogeny has the functional domains of histone acetyltransferases and firmly established their distinct taxonomic position as a their functions, type A HATs can be further classified specific group of eukaryotes belonging to the phylogenetic into different families, such as GNAT, Elp3, Hpa2, MYST, lineage of biflagellate “heterokont” organisms universally p300/CBP, TAFII250 and ACTR/SRC-1 [25]. Type B HATs referred to as “Stramenopila”, with photosynthetic algae are located in the cytoplasm, where they can acetylate free such as brown algae and diatoms [39]. Stramenopiles and histones prior to assembly into nucleosomes, such as Hat1 Alveolates, which include the apicocomplexa, ciliates and in yeast [26]. Each family of HATs has its own highly dinoflagellates, compose the superkingdom Chromalveo- conserved sequence [27]. lates [40–43]. However, there are barely any data available HDACs are divided into four phylogenetic groups, on the genes and the role of epigenetic modifications in Class I, II, III and IV, according to localization and oomycetes, or even in the Stramenopiles. tissue-specific expression [28]. In addition, HDACs can Considering the importance of histone acetylation in be classified into two types based on their dependence epigenetic modifications and the existence of diverse his- on zinc or NAD co-factors [29, 30]. The Class I, II and tone acetyltransferases and deacetylases in many eukaryote IV HDACs are zinc-dependent, while the Class III species investigated, we postulated that species in oo- HDACs are NAD-dependent. mycetes have diverse histone acetyltransferases and dea- Previous studies have found that the core domain of cetylases. With the available genome sequences of several HATs plays an especially important role in histone oomycetes species, we investigated the candidate genes of substrate catalysis, while the N- and C-terminal domains histone acetylation in ten sequenced species and provide a are key elements in histone substrate binding [31]. comprehensive overview of the structure, diversity, phyl- Analysis of acetylated lysines on histone revealed that ogenyandtheexpressionpatternofHATs and HDACs of the region of histone acetyltransferase domains inte- oomycetes in this study. racting with peptide substrates typically do not exceed 14 to 20 amino acid (aa) in length flanking each lysine Methods [31] (7 to 10 residues on each side). In addition, an ana- Oomycetes for database searches lysis of frequency distribution of amino acids surround- Genomesoftenspeciesofoomyceteswithdivergentlife ing acetylated and non-acetylated lysines in histone styles and belonging to various taxa in oomycetes were indicated that residues surrounding acetylated lysine used. They included the pathogen of fresh water fish, S. were usually small and polar, while basic, acidic and parasitica in Saprolegniaceae of Saprolegniales; the soil- hydrophobic residues were more inclined to surround borne plant pathogen Pythium ultimum in Pythiaceae of non-acetylated lysine [31]. Based on these findings, Basu Pythiales; the soil-borne plant pathogens Phytophthora cap- et al. [32] developed the program PredMod for predict- sici, P. cinnamomi, P. infestans, P. parasitica, P. ramorum, ing the acetylated sites. and P. sojae, and the obligate plant parasite Hyaloperonos- The Oomycetes are a group of fungus-like eukaryotes pora parasitica in Peronosporales; and the air-borne obli- with diverse microorganisms living in marine, fresh- gate plant parasite Albugo laibachii in Albuginaceae of water, and terrestrial environments [33, 34]. Many mem- Albuginales (Fig. 1). Other than H. parasitica and A. laiba- bers of oomycetes are important pathogens of plants chii, the rest were facultative parasites. Wang et al. BMC Genomics (2016) 17:927 Page 3 of 14

Total HATs HDACs numbers

Abluginales Albugo laibachii 23 Plants

Obligate Hyaloperonospora parasitica 30 parasites

Phytophthora infestans 44

Phytophthora capsici 46 Plants Peronosporales Phytophthora ramorum 41

Phytophthora sojae 44 Soil

Phytophthora parasitica 56

Facultative parasites Phytophthora cinannmomi 53

Pythiales Pythium ultimum 48

Fresh Saprolegniales Saprolegnia parasitica 60 water 0 10203040500 6 12 18

Gcn5 Elp3 Hpa2 TAFII250 Class I Class II P300 MYST Hat1 putative novel HAT Class III Fig. 1 Numbers of HATs and HDACs found in ten species of oomycetes. Columns in different colors represent different families of HATs and HDACs. Species in brown are soil-borne plant pathogens

Gene identification and analysis of HATs and HDACs peptides were predicted using the CBS Prediction Gene sequences and proteomes of oomycetes species used in Servers (http://www.cbs.dtu.dk/services/). The second- this study were retrieved from the database of the BROAD ary structures of proteins were predicted using the online INSTITUTE (https://www.ncbi.nlm.nih.gov/bioproject), the program Psipred (http://bioinf.cs.ucl.ac.uk/psipred/) [55] DOE Joint Genome Institute (http://www.jgi.doe.gov/), the and CFSSP (http://www.biogem.org/tool/chou-fasman/) EnsemblGenomes (http://www.ensemblgenomes.org/) and [56]. The sequences accession numbers and features are the Pythium Genome Database (http://pythium.plantbio- listed in Additional file 1. Sequence logos were created logy.msu.edu/index.html) [44–50]. Additional searches for with WebLogo (http://weblogo.berkeley.edu/logo.cgi) for genes of diatoms (Phaeodactylum tricornutum, Thalassio- displaying the conserved peptides of motif A in the HATs sira pseudonana), ciliates (Tetrahymena thermophila), of oomycetes [57]. diplomonads (Giardia lamblia), yeast (Saccharomyces cerevisiae), human (Homo sapiens), Arabidopsis (Arabidopsis Sequence alignments and phylogenetic analysis thaliana) and other species were retrieved from the NCBI, To infer the phylogenic history of oomycetes genes, we UniProt, and KEGG databases (Additional file 1). All compared the oomycetes genes with their orthologs in the sequences were checked for annotation mistakes diatoms (Ph. tricornutum, T. pseudonana), diplomonads using the following methods. The structural domains of (G. lamblia), ciliates (Te. thermophile), yeast (S. cerevi- protein sequences were predicted using the SMART siae), human (H. sapiens), Arabidopsis (A. thaliana), and (http://smart.embl-heidelberg.de/) and Pfam (http:// some other related species retrieved by BLAST against pfam.sanger.ac.uk/) databases and NCBI CD (Conserved the genome databases with HATs and HDACs in oomy- Domains) - Search (http://www.ncbi.nlm.nih.gov/Struc- cetes (e value <1e-10) (Additional file 1). The amino acid ture/cdd/wrpsb.cgi). Additional domains were predicted sequences of conserved core domains were pairwise and and verified with the InterProScan (http://www.ebi.ac.uk/ multiple aligned using ClustalW2 (http://www.ebi.ac.uk/ Tools/pfa/iprscan/) and ExPASy Prosite (http://prosite.- Tools/msa/clustalw2/) [58]. In Pairwise Alignment, expasy.org/) [51–54]. The combinations of domains in the BLOSUM62 with a gap extension of 0.1 and 0.2 was HATs were BLAST against the databases KEGG and used as the protein weight matrix and in Multiple UniProt with default parameters (e value <1e-10) to search Alignment, respectively. The resulted sequence align- for their homologs in the genomes of other species. Signal ments were used to construct phylogenetic trees with Wang et al. BMC Genomics (2016) 17:927 Page 4 of 14

the maximum likelihood evolution algorithm in MEGA To obtain biological material at different stages of sexual 5.22 [59]. A Poisson correction was used for multiple development, a polycarbonate membrane with 0.4 μm substitution models and pairwise deletion was used for pores (Millipore, Ireland, diameter 43-mm) was placed on gap split data treatment. The statistical strengths were the surface of a rye tomato agar plate (60 mm), then, iso- assessed by bootstraps with 1000 replicates or replica- lates YZ-6 (A1) and 80787-94L (A2) were incubated 2 cm tions. To investigate the events of gene duplication and apart from each other on the polycarbonate membrane in loss happened during evolution of oomycetes, we con- each plate. These plates were then incubated at 18 °C in structed a phylogenetic tree of the ten species of oomy- darkness. After 4, 10 and 14 days, mycelia or mycelia with cetes in this study with two diatom species (Ph. oospores were harvested from the junction between the tricornutum and T. pseudonana) as out groups using the two isolates. Meanwhile mycelia were collected similarly Composition Vector Tree (CVTree) based on proteomes from the single culture of A1 and A2 isolates, respectively [60]. The statistical strengths of the topology were and used as controls. assessed by bootstraps with 100 replicates. Then, we After grinding the samples with liquid nitrogen the reconcile the HATs and HDACs ML trees with the spe- total RNA was extracted using NucleoSpin RNA Plant cies phylogeny using NOTUNG (v2.6; 1.5 duplication kit (MACHEREY-NAGEL, Düren, Germany). The quan- and 1 loss cost) [61, 62]. The weakly supported branches tity and quality of RNA were measured with a Thermo (<80% bootstrap values) were rearranged to minimize Scientific NanoDrop 2000 spectrophotometer (Thermo duplication/loss costs (D/L scores), and orthologous Fisher Scientific Inc., Wilmington, USA). groups were formed based on duplications at the least common ancestor (Additional file 2). Expression analysis of HATs and HDACs in Phytophthora infestans Prediction of histone acetylation sites In order to gain insight into the possible role of predicted For each lysine of histone, probabilities of acetylation were oomycetes HATs and HDACs, we examined the expres- predicted with the PredMod (http://ds9.rockefeller.edu/ sion levels of 33 HATs and 11 HDACs genes of P. infestans basu/predmod.html) [63] with the setup value of 12 as the through SYBR green real-time qPCR assay with specific maximum number of residues flanking each lysine. primers designed in this study (Additional file 3). For Sequence logos were created with WebLogo (http:// cDNA synthesis, 1 μg of total RNA was reverse transcript weblogo.berkeley.edu/logo.cgi) for displaying the flanking with oligo(dT)18 primer using the Reverse Transcriptase residue distribution of the predicted acetylated and non- M-MLV (TaKaRa Bio Inc. Shiga, Japan) following the acetylated lysine on histone N-terminal tails [57]. manufacturer’s instructions. Before performing the real- time qPCR, the specific primers were used to amplify the Preparation of biological material and RNA extraction target genes. The products yield were then sequenced and P. infestans, MX5-1 (A1) and YZ-6 (A1) from potato in aligned with the target genes to ensure that the products China and 80787-94L (A2) from USA [64] were used in were from the target genes. The amplified sequences this study. The biological materials were obtained at that longer than 200 bp were submitted to NCBI Gen- various stages of asexual development by growing the Bank data library under accession numbers KX492573 isolate MX5-1 on 150 tomato rye agar plates (90-mm) to KX492582 (Additional file 4). Amplifications were and were kept at 18 °C in darkness for 10 days [64]. conducted in 25 μl volume containing 50 ng cDNA, Sporangia (SP) sample was harvested by washing mycelia 0.4 μMeachprimer,0.2mMdNTP,1UrTaqDNA mats with sterile water, accumulated all from 30 plates polymerase (TaKaRa Bio Inc. Shiga, Japan), and 17.3 μl then, was centrifuged for 8 min at 2,000 rpm. The spor- of sterilized distilled H2O with the reaction conditions angia suspension of other 120 plates was placed at 4 °C as follows: 95 °C for 4 min, followed with 30 cycles of for 2 to 3 h to induce zoospores (ZO) formation and it 95°Cfor30s,60°Cfor30s,and72°Cfor30s,atlast was divided into four samples each consists of 30 plates 72 °C for 4 min. SYBR green real-time qPCR were as collected by different ways. The 1st ZO sample was performedinanABI7500detectionsystem(Applied collected by centrifuge the suspension at 2,000 rpm for Biosystems, Foster City, CA, U.S.A.). Amplifications 8 min. The 2nd sample of zoospores were induced to were conducted in 20 μl volume containing 10 ng form cysts (CY) by vortex at 40 s and used as CY cDNA, 0.2 μM each primer (Additional file 3), 1× ROX sample. Whereas the 3rd Zoospores sample was incu- Reference Dye II, 1× SYBR Premix Dimer Eraser bated in pea broth at 120 rpm, at 18 °C for 3 h and the (TaKaRa Bio Inc. Shiga, Japan), and 4.8 μl of sterilized geminated cysts (GC) were collected by centrifuge at distilled H2O with the reaction conditions as follows: for 2,000 rpm for 8 min as GC sample. The last zoospore calculate Ct values, 95 °C for 30 s, followed with 40 cycles sample was grown on pea broth at 120 rpm, at 18 °C in of 95 °C for 5 s, 60 °C for 34 s; and for obtain melt curves, darkness for 48 h, and used as MY samples. 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s. The Wang et al. BMC Genomics (2016) 17:927 Page 5 of 14

gene of elongation factor 1 (ef1, PITG_06722.1) in P. infes- the AT_1 domain, but a few also have a signal peptide at tans was used as an inner control. The whole experiment their N-terminal region, similar to some Hpa2s in diatoms was repeated with two different sets of biological samples. (Fig. 2). Only one protein of the HAFs family, TAFII250, The 7500 system sequence detection software was used was recognized in most oomycetes species, except in Py. for data analysis, and the heatmaps and the HCL trees ultimum. The approximately 500 aa long HAF consists of were constructed using MultiExperiment Viewer (v. 5.0). an AA_kinase (PF00696) domain at the N-terminus and As same color can mean different expression values on an AT_1 domain at the C-terminus (Additional file 6). different genes, comparison of colors only justified be- Two to four HACs that are orthologs of the p300/CBP tween expression levels of different development stages of histone acetyltransferase family are encoded in each thesamegene. oomycete genome. Their domain compositions are vari- We also analyzed the EST, cDNA, TPM and the able in the presence of the ZnF-TAZ (SM000551) domain. expression profiles data of P. infestans from previous Most of them contain a KAT11 (SM001250), a BROMO studies (Additional file 5). These data were retrieved (PF00439) and a PHD (SM000249) domain (Additional from the NCBI UniGene website, and Gene Expression file 7). KAT11 is an ortholog of the fungal KAT11 protein Omnibus DataSets [65, 66]. The expression values were (previously known as RTT109), which is required for log2-transformed and used to construct the heatmap. H3K56 acetylation [68]. Besides, most oomycetes encode one MYST acetyltransferase in the HAM family, except Results and discussion for S. parasitica, which has two. HAM is approximately Identification of HATs and HDACs in oomycetes 460 aa long and is recognized by the N-terminal Chromo Through a domain-based search and the analysis of the (PF00385) domain and the C-terminal 200 aa MOZ-SAS structure and core domain in comparison with the HATs (PF01853) domain (Additional file 8). Moreover, all of the and HDACs in reference species, we identified many oomycetes species analyzed here has a homologous pro- diverse HATs and HDACs in each species, including tein of the Hat1_N family, namely Hat1. The Hat1 protein some putative novel HATs. There are 18 and 20 HATs contains a conserved Hat1_N (PF10394) domain, which is in the two obligate plant parasites, A. laibachii and H. the same as the N-terminal part of the AT_1 domain of parasitica, respectively, and from 24 to 44 HATs in 8 histone acetyltransferase. As an exception, the S. parasi- facultative parasites (Fig. 1). There are 5 HDACs in A. tica Hat1 (XM_012343809.1) carries an additional AT_1 laibachii and 10 in H. parasitica, and from 10 to 17 in domain similar to AthHat1 (NM_125057.3) in Arabidopsis eight facultative parasites. In general, more HATs and (Additional file 9). HDACs were found in the facultative parasitic oomy- During our investigation, three novel families of HATs cetes than in the obligate parasitic ones. were revealed in oomycetes through combinations of domains. All three have a common functional AT_1 Histone acetyltransferases in oomycetes domain; as illustrated, PifHAT1 and its homologs (I) also Both Type A and Type B HATs were found in oomy- contain an extra N-terminal LCM (PF04072) domain. cetes in five families, comprising HAGs, HAFs, HACs, Similarly, PifHAT2 and its homologs (II) have a Fascin HAMs, and Hat1s (Fig. 1). Unexpectedly, we also discov- (cl00187) and a DUF706 (PF05153) C-terminal domain, ered three putative novel families of HATs with new while PifHAT3 and its homologs (III) have an additional combinations of conserved domains. The HAG group C-terminal PhzC-PhzF (PF02567) domain (Additional consists of Gcn5, Elp3 and Hpa2. Gcn5 is recognized in file 10). Such joint synchronization has not been re- most oomycetes species, except P. ramorum (Fig. 1). It ported previously and we did not detect additional contains two conserved domains: a histone acetyltrans- homologs other than one PifHAT3 (XM_002895006.1) ferase domain (Acetyltransf_1 (AT_1) PF00583) of 90 aa in the genome of T. pseudonana, a diatom. In addition, at the N-terminus, and a bromodomain of 110 aa 2–3 putative novel HATs were found in most facultative (PF00439) at the C-terminus [67] (Fig. 2). Sequence parasites; while only one was identified in each of the alignment reveals that oomycetes Gcn5 is highly con- two obligate parasites H. parasitica and A. laibachii. served with 72.4% identity. Two proteins from the Elp3 Secondary structural predictions suggest that most of family, Elp3-1 and Elp3-2, were recognized in most HATs have conserved motifs in its conserved domain. oomycete species, although only one was found in P. Except Hat1, oomycetes HATs have a conserved core ramorum and H. parasitica. These proteins have an region, including motif D, A and B, for the function of AT_1 domain at the C-terminus and a 260-aa Elp3 histone acetyltransferase (Fig. 3). In addition, a motif C domain (SM000729) at the N-terminus. Unlike the Gcn5 is present at the N-terminus of protein Gcn5s, Hpa2s, and the Elp3 families, Hpa2 family has multiple mem- HAFs, and Hat1s. Furthermore, the motif A has a bers, ranging from 9 in A. laibachii to 29 in P. parasitica conserved peptide Gln/Arg-X-X-Gly-X-Gly/Ala, which and S. parasitica (Fig. 1). Many Hpa2 proteins have only associates with acetyl-CoA recognition and binding. Wang et al. BMC Genomics (2016) 17:927 Page 6 of 14

22GAHac

PpaHAG20AlaHAG10

SceELP3 SpaHAG32 HsaELP3 SpaHAG28TpsHAG8 AthELP3

PsoHAG25 P PpaHAG27 PsoHAG22 PifHAG23 PcnHAG25 SpaHAG21 SpaHAG23 PyuHAG27 PcnHAG28SpaHAG11 AlaHAG12 PtiHAG10 PsoHAG23 TpsHAG11 TetHAG18 GlaELP3 SpaHAG10 PcaHAG27PpaHAG5 AlaHAG9 PyuHAG28 PcaHAG26 HpaHAG11 PsoHAG20 PcnHAG29 PpaHAG8 PrHAG14 PcnHAG27 PcaHAG9 SceNAT5 PcaHAG25 39 PpaHAG23 PifHAG22 PcaHAG24 58 SpaHAG18 SpaHAG8 94 58 SpaHAG5 AlaHAG11 79 SpaHAG4 96 45 SpaHAG7 PyuHAG5 SpaHAG19 PifHAG17 79 SpaHAG9 62 SpaHAG12 PcaHAG23 93 40 74 99 TetHAG17 SpaHAG16 PsoHAG21 31 PrHAG5 47 SpaHAG27 67 SpaHAG29SceNAT4 PpaHAG2 32 PtiHAG7 PcnHAG26 50 55 HsaCSRP2BP AthHAG19 51 PtiHAG5AthHAG6 95 SpaHAG25 52 SpaHAG3HsaGNA1 PtiHAG3 46 45 46 AthHAG10 TpsHAG3 45 TetHAG11 48 39 GlaHAG5SceGNA1 HsaNAT9 37 AthHAG17 86 69 30 GlaHAG6 SpaHAG1 37 TetHAG6 PsoHAG11 56 100 TetHAG16AthHAG2 PcnHAG3 35 AthHAG20 30 88 AthHAG3 PrHAG4 36 PpaHAG30 46 46 AthHAG21 44 91 PifHAG6 TetHAG14AthHAG7 84 62 32 57 36 PsoHAG4 60 37 TpsHAG5 PcnHAG23 AthHAG12 PifHAG15 TetHAG15 PpaHAG29 71 TetHAG13 PcaHAG3 97 TetHAG7 88 42 TpsHAG10 PyuHAG6 97 52 83 SpaHAG17 69 TpsHAG9HsaNAT8L HsaNAT8 HsaNAA50TetHAG2 62 HsaNAT8B AthHAG13 77 AthHAG5 SpaHAG2 32 SpaHAG20 PpaHAG12 34 72 81 PtiHAG8 PifHAG12 PifHAG21 PsoHAG3 76 PpaHAG17 10048 77 PcaHAG4 37 46 PcaHAG11 PcnHAG20 HpaHAG7 PcnHAG6 TetHAG5 PcnHAG7 TetHAG3 95 PifHAG16 TetHAG4 47 79 36 43 HsaSNAT HsaNAT15 99 51 SpaHAG26 AthHAG26 34 ScePAA1 AthHAG4 96 HsaSAT2 AlaHAG2 HsaSAT1 SpaHAG15 PtiHAG6 HpaHAG2 92 AthHAG18 PpaHAG1 AthHAG22 68 PrHAG9 PifHAG2 30 76 91 PrHAG10 PrHAG1 46 46 PsoHAG17 PcaHAG2 89 44 38 PifHAG11 PsoHAG2 73 96 PcaHAG19 PcnHAG21 30 58 PcnHAG19 39 99 100 PpaHAG19 AlaHAG5 SceHPA2 AlaHAG4 SceHPA3 PyuHAG16 34 32 TetHAG12 49 SpaHAG13 HpaHAG10 TpsHAG4 PpaHAG13 49 55 100 HsaNAT6 PifHAG19 38 32 PpaHAG6 PcaHAG21 69 51 33 PpaHAG7 PsoHAG19 5469 56 PifHAG18 PcnHAG24 34 70 PpaHAG21 PsoHAG18 64 41 95 PpaHAG24 PcnHAG22 69 PifHAG20 PrHAG11 55 98 PcaHAG28 70 PpaHAG4 30 PcaHAG16 PcaHAG20 66 5580 PcaHAG14 PifHAG13 PcaHAG17 HpaHAG9 87 35 PcnHAG12 PsoHAG12 AthHAG23 65 PsoHAG16 PyuHAG17 69 59 PcnHAG14 AlaHAG8 95 93 76 PpaHAG26 TpsHAG7 35 PpaHAG32 SpaHAG14GlaGCN5 97 PcnHAG9PpaHAG31 PsoHAG14 HsaKAT2B 58 33 PpaHAG28 PrHAG8 HsaKAT2A 34 SceGCN5TetHAG1 47 58 34 PcnHAG8 5048 PcaHAG12 AthGCN5 PifHAG9 SpaHAG30 PpaHAG3 PyuHAG29 PyuHAG9 AlaHAG1 83 PyuHAG12 PrHAG6 92 45 47 PyuHAG19 62 PyuHAG1 PpaHAG10 38 PcaHAG1 92 PyuHAG24 98 31 PyuHAG8 HpaHAG1 PifHAG1 PcaHAG18 32 57 PsoHAG15 1 PsoHAG1 30 96 PcnHAG13 PcnHAG4 59 52 PyuHAG25 5393 5 PyuHAG26

PyuHAG10 5 PyuHAG7 100 89 AlaHAG7 87 PyuHAG3 30 56 PyuHAG4 PrHAG2 64 5235 38 44 PyuHAG15 PrHAG13 PyuHAG20 87 32 61 46 PyuHAG2 PsoHAG5 77 51 PyuHAG13 PcnHAG16 91 PpaHAG25 PcaHAG5 85 PcnHAG10 PifHAG7 88 30 72 30 65 5083 PcaHAG13 PifHAG3 HpaHAG8 82 PpaHAG15 PsoHAG7 82 PcaHAG15 44 94 PpaHAG11

PpaHAG33 47 PcnHAG11 PcnHAG18 PsoHAG15 2 PyuHAG22 80 30 33 PyuHAG21 41 HpaHAG3 AthHAG24 30 GlaHAG3 AthHAG25 55 PpaHAG14 40 AthHAG14 PifHAG10 PtiHAG4 AthHAG15 PsoHAG8 51 TetHAG10 66

56 84 SceMAK3

PcnHAG15 42 PyuHAG23 95 HsaNAA30 SpaHAG24 PyuHAG11 AlaHAG3 PyuHAG14 AthHAG16 PsoHAG6 PyuHAG18 HpaHAG6 PcnHAG5 PcnHAG17PrHAG12 PcaHAG10 PifHAG8 PrHAG7 PcaHAG6 PpaHAG22 PsoHAG13 PpaHAG18PifHAG14HpaHAG12 AT_1 SceNAT3

GlaHAG4 9

TetHAG8 9 Gcn5 G

HsaNAA20TpsHAG1 PtiHAG1 AHht AthHAG11 A

PifHAG4 PtiHAG9

PpaHAG9 PrHAG3 TpsHAG6 H BROMO SpaHAG31

HpaHAG5 PifHAG5 PcnHAG1

PcnHAG2 eT PsoHAG9 PcaHAG8

PcaHAG7 AthHAG8

PtiHAG2

AlaHAG6

PsoHAG10 A

GlaHAG2 HpaHAG4

SceARD1 SpaHAG6

TpsHAG2 PpaHAG16 HsaNAA10 HsaNAA11 Elp3 Hpa2 SP Elp3 PCAF_N Elp3-1 Elp3-2 Fig. 2 Phylogenetic tree of HAGs in oomycetes. The maximum-likelihood tree was constructed with sequences of conserved domain AT_1 of HAGs. Bootstrap values (≥30%) are shown near the tree nodes. The predicted HAGs were attributed to three known families, Gcn5, Hpa2 and Elp3. The conserved domains of each family were highlighted by different colors. In oomycetes, the Gcn5 family protein (the branch colored red) is composed of AT_1 and Bromodomain. The Elp3 family (the branch colored blue) encodes an Elp3 domain at the N-terminal in addition to the AT_1 domain, while most Hpa2s have only the AT_1 domain. Reference species are highlighted in different colors. Pif, Phytophthora infestans; Pr, P. ramorum; Pso, P. sojae; Pca, P. capsici; Ppa, P. parasitica; Pcn, P. cinnamomi; Pyu, Pythium ultimum; Al, Albugo laibachii; Hpa, Hyaloperonospora parasitica; Spa, Saprolegnia parasitica; Hsa, Homo sapiens; Sce, Saccharomyces cerevisiae; Ath, Arabidopsis thaliana; Pti, Phaeodactylum tricornutum; Tps, Thalassiosira pseudonana; Tet, Tetrahymena thermophile; Gla, Giardia lamblia

However, like the KAT11 domain of HACs in human seven members occurred in each species. The protein and Arabidopsis [27], no conserved peptide was found structures are simple containing only the conserved core in motif A of KAT11 of oomycetes. Similar to the func- domain, the Hist_deacetyl (PF00850) of histone deacety- tional Hat1p (NM_001183815.1) in Saccharomyces cere- lases (Fig. 4). For class II HDACs of oomycetes, two to visiae [69], the Hat1_N domain of oomycetes Hat1s seven members were found in each species. With the contains only motifs C and D. Hist_deacetyl domain these members have in common, some contain new conserved domains that have not Histone deacetylases in oomycetes been documented in the known histone deacetylases, Only three Classes (I, II & III) of HDACs are identified such as a repeat of 3 to 4 ANK domains (SM000248), in oomycetes (Figs. 1 and 4). For Class I HDACs, two to which could form a helix-loop-helix structure involved Wang et al. BMC Genomics (2016) 17:927 Page 7 of 14

the HAT core HATs Conserved domain N C D A B C 266 293 312 335 366 388 400 Gcn5 AT_1 1 2 3 4

522 555 600 620 640 Elp3 AT_1 18 19

18 38 85 110 155 180 200 Hpa2 AT_1 1 2 3 4

407 424 447 465 503 520 TAFII250 AT_1 10 11 12 13

510 540 585 635 p300/ KAT11 12 13 CBP 296 317 348 380 MYST MOZ-SAS 3 4

82 105 134 160 Hat1 Hat1_N 3 4

putative AT_1 novel

Fig. 3 Conserved motifs identified in different families of HATs in oomycetes. Motif A of most of the HATs identified has a highly conserved peptide Arg/Gln-X-X-Gly-X-Gly/Ala. The overall height of the stack predicted by the WebLogo indicates the sequence conservation at each amino acid, while the height of symbols within the stack indicates the relative frequency of each amino acid at that position in protein-protein interaction (Fig. 4), or a combination of a including PHD, RING and ZnF-ZZ in addition to the com- C-terminal Med27 domain (PF11571) and a N-terminal monSir2domain(PF02146).OnetosixmembersofClass cascade containing a PHD domain (SM000249) along with III HDACs are identified in each species of oomycetes. two AP2 domains (SM000380). The Med27 domain (PF11571) is related to regulation of the transcriptional ac- Phylogenetic and evolutionary analysis of HATs and tivity of RNA polymerase II. The PHD domain (SM000249) HDACs in oomycetes is a C4HC3 zinc-finger-like motif thought to be involved in To investigate the evolution history of HATs and HDACs epigenetics and chromatin-mediated transcriptional regula- protein families in oomycetes, we compared them with tion, and the AP2 domain (SM000380) is related to DNA orthologs of reference species including diatoms, ciliates, binding. The presence of various domains on a protein indi- diplomonads, yeast, human, Arabidopsis and some other cated that these proteins could form complexes with other species in genome database. Phylogenetic analysis indi- proteins. Similar situation was also found in some of the cates that different groups of oomycetes HATs have differ- Class III HDACs, which contain two of the three domains ent evolution history. Wang et al. BMC Genomics (2016) 17:927 Page 8 of 14

PpaSir2.1 PcaSir2.1

PsoSir2.1PifSir2.1

PcnSir2.2 PcnSir2.1 PrSir2.1 HpaSir2.1 PyuSir2.2

PsoHDAC4 PcaHDAC6 PcnHDAC2 PcnHDAC1 GlaSir2.1 HpaHDAC1 PrHDAC1 GlaSir2.2 AlSir2 PpaHDAC1 PifHDAC1 AlHDAC3 PrSir2.2 55 PpaHDAC2 PifSir2.2 PcnHDAC3 PpaSir2.2 PcaHDAC8 HpaSir2.2 53 PifHDAC6 52 PrHDAC2 PcnSir2.3 PifHDAC8 PsoSir2.2 PyuHDAC1 SpaHDAC1 PcaSir2.3 PyuHDAC2 PyuSir2.1 51 SpaHDAC6 SpaSir2.1 SpaHDAC2 SceHOS2 SceSIR2 93 SceRPD3 SceHST1 56 HsaHDAC3 91 AthHDA9 SceHST2 55 PtiSIR2.1 AthHDA7 100 AthHDA6 HsaSIRT3 94 HsaSIRT2 TetHDAC1 100 78 TetHDAC4 TetSir2.3 TetHDAC3 TetSir2.2 92 PsoHDAC2 TetSir2.11 PrHDAC9 52 62100 PifHDAC3 TetSir2.1 58 HsaSIRT1 PcaHDAC7 PpaHDAC3 SceHST4 50 HpaHDAC3 SceHST3 81 HpaHDAC2 GlaSir2.3 PcnHDAC4 69 PcaSir2.2 69 AlHDAC4 PpaSir2.3 99 PyuHDAC3 63 SpaHDAC4 PcnSir2.4 87 SpaHDAC5 56 PrSir2.6 100 SpaHDAC3 PrSir2.5 94 PtiHDAC1 HpaSir2.5 66 83 TpsHDAC1 PyuSir2.3 70 8059 HsaHDAC1 HsaSIRT4 HsaHDAC2 AthSRT2 HsaHDAC8 PtiHDAC2 TpsSIR2.1 98 93 AthHDA19 PrSir2.3 TetHDAC2 PrSir2.4 SceHOS1 PyuSir2.4 83 100 SpaHDAC7 PcnSir2.5 PyuHDAC4 88 PifSir2.3 PrHDAC3 57 PpaHDAC8 PsoSir2.3 91 93 95 PpaHDAC5 PpaSir2.4 PcnHDAC7 81 HpaSir2.3 62 GlaHDAC1 SpaSir2.2 TetHDAC6 AthSRT1 TetHDAC5 100 HsaSIRT6 88 PtiHDAC3

53 AthHDA15 HsaSIRT7 77 TetSir2.6 AthHDA5 59 AthHDA18 TetSir2.4 HsaHDAC7 Hist-deacetyl TetSir2.5 98 81 56 HsaHDAC5 75 TetSir2.7 74 HsaHDAC4 95 77 95 71 HsaHDAC6.2 HsaSIRT5 53 Sir2 GlaSir2.5 9990 64 HsaHDAC10 60 HsaHDAC6.1 TpsSIR2.2 81 PtiHDAC4 PtiSIR2.2 SceHOS3 ANK 52 82 SpaHDAC12 TetSir2.10 99 TetSir2.9 79 68 PrHDAC7

60 85 PsoHDAC6 75 Med27 PpaHDAC6 TpsHDAC3 PcaHDAC4 PtiHDAC7 100 PcnHDAC6 PifHDAC7 SpaHDAC9 59 HpaHDAC4 ZnF-ZZ 64 PyuHDAC6 PcaHDAC3 98

78 SpaHDAC10 86 AlHDAC2 PsoHDAC3 TpsHDAC4 PtiHDAC6 Znf-UBP PrHDAC4 AthHDA14 PpaHDAC10 SpaHDAC8 PyuHDAC5 PcaHDAC5 AlHDAC1 PsoHDAC1 PcnHDAC8 PcnHDAC5 PrHDAC6 PrHDAC8 PifHDAC4 PpaHDAC4 HpaHDAC5 SP PifHDAC2 5C

SpaHDAC14TpsHDAC5 A

DH PHD SpaHDAC11PsoHDAC7

PpaHDAC7 f PcnHDAC10 i PcaHDAC1PrHDAC10

PyuHDAC7

SpaHDAC13 PpaHDAC9 PrHDAC11 PcnHDAC9 PsoHDAC5 PcaHDAC2 RING Arb2 Class I Kelch-1 Class II DUF592 Class III AP2 Fig. 4 Phylogenetic tree of HDACs in oomycetes. A maximum-likelihood phylogenetic tree was constructed with sequences of HDAC conserved domains from the species described in Fig. 2. All the predicted HDACs were attributed to three known classes (I, II and III) based on the combination of domains. Conserved domains of each family and reference species were highlighted by different colors and bootstrap values (≥50%) are shown near the tree nodes

Gcn5s of oomycetes are clustered in one clade, together Gcn5 and Py. ultimum HAF in the genome database is with the orthologs of reference species, including human, probably due to the deficiencies in gene assembly as Gcn5 Arabidopsis, yeast, diatoms, diplomonads (Giardia lam- (PYU1_T008661) of Py. ultimum was also absent in the blia) and ciliates (Tetrahymena thermophila) (Fig. 2) with earlier version of genomic data. high supporting value, indicating that Gcn5s are evolu- Unlike oomycetes Gcn5s and HAFs families, phylogenetic tionary highly conserved proteins of ancient lineage pre- analysis showed that members of Hpa2 proteins in each dating speciation in oomycetes. HAFs of oomycetes oomycetes species are split into several clades with othologs are clustered in one clade that close related to the of different reference species (Fig. 2). Some (tagged with a clade containing diatoms and red algae (Galdieria red square in Fig. 2) are homologs of experimentally sulphuraria, Chondrus crispus and Cyanidioschyzon verified AthHAG26 (NM_111168.1, tagged with a black merolae) (Additional file 6). This result indicated that triangle in Fig. 2). Some (tagged with a blue square) are oomycetes HAFs were obtained in ancient times before homologs of SceNAT3 (NM_001184228.1), AthHAG16 speciation in oomycetes, and they had a common ancestor (NM_129369.5) and HsaNAA11 (NM_032693.2) (tagged with that of red alga and diatoms. The lack of P. ramorum with a black square in Fig. 2), which are acetyltransferases Wang et al. BMC Genomics (2016) 17:927 Page 9 of 14

at the protein level. Some (tagged with a green square clustered in a region approximately 10 Kb on the gen- in Fig. 2) are homologs of the predicted acetyltrans- ome of Py. ultimum. These cases indicating that duplica- ferases AthHAG19 (NM_128762.1) and AthHAG23 tion of HATs and HDACs happened after speciation in (NM_148472.2) (tagged with an empty black square oomycetes. We also found the aggregation of members of in Fig. 2). These results suggested that the Hpa2 gene HATs with various identities on the genome, such as a clus- in oomycetes derived from different lineages in an- ter of four genes (SpaHAG4 (XM_012338354.1), SpaHAG7 cient times. Similar cases were also found in HACs, (XM_012338355.1), SpaHAG19 (XM_012338350.1) and HAMs and HDACs of oomycetes (Additional files 7 SpaHAG9 (XM_012338349.1)) in a region of approximately and 8, and Fig. 4). HACs in oomycetes were split into 10KbinthegenomeofS. parasitica (Additional file 11b), four clades. Clades I and II are closely related to diatoms, suggesting that duplication and divergence of these but the other two Clades (III and IV) do not have any genes happened after speciation in oomycetes. Similar othologs in reference species, or in the current genome cases were also found commonly in the facultative database (Additional file 7). For HAMs, most were species such as Py. ultimum and P. capsici, but not grouped into a clade (oomycetes I in Additional file 8), in obligate parasitic species. with one ortholog of diatoms (XM_002176539.1) and To investigate the frequency of gene duplication and some orthologs in marine algae belonging to Strameno- loss of HATs and HDACs in oomycete evolution, we piles, disclosing their common ancestral origin. The other first constructed a phylogenetic tree of the ten oomy- copy of HAM (XM_012345245.1) in S. parasitica was cetes with closely related diatom, and then reconciled grouped into a different clade with HAMs of water molds the HAGs and HDACs ML trees with the predicted spe- and another copy of HAM in diatoms (XM_002176731.1). cies phylogeny tree. The obtained species phylogeny tree For class I HDACs, all of these oomycetes species have at is highly supported with bootstrap values ≥99% for all least two members, and phylogenetic analysis showed that nodes. It is consistent with the known evolution trends one is a homolog of diatoms; and the other is a homolog of oomycetes, clearly separating the Saprolegniales, of yeast (Fig. 4). Likewise, most class II and III HDACs of Pythiales, Peronosporales, and Albuginales [70], as well oomycetes were homologs of diatoms, ciliates and diplo- as the six species in clades 1, 2, 7, and 8 of Phytophthora monads, respectively (Fig. 4). [71]. Both duplication and gene losses were found in Except the situations descried above, phylogenetic ana- HATs and HDACs of most species. In comparison with lysis showed that the Elp3-1 protein (one star in Fig. 2) facultative plant parasitic species living in soil and plant, is clustered with orthologs in diatoms, yeast, human and obligate plant parasitic species experienced less gene du- Arabidopsis, whereas the Elp3-2 protein (double stars in plication and more gene loss events, ending up with a Fig. 2) is assembled in a separate clade without homo- contraction of HATs and HDACs in their genomes logs in any reference species. As Elp3-1 and Elp3-2 (Fig. 5). The facultative parasitic oomycetes of fresh proteins are group in the same super clades with high water fish, S. parasitica had the highest frequency (12) support value, the two Elp3 genes in each species are of gene duplication, but no gene loss (Fig. 5), gaining the likely originated from the same ancient lineage, but a most amounts of HATs and HDACs in its genome. Vari- divergence happened later in ancient time, at least before ation in frequency of gene duplication and loss were also the speciation of these species in oomycetes (Fig. 2). observed among the 6 species of Phytophthora. Within Regarding the putative novel HATs identified in oomy- the same clade, the species found more frequently in cetes based on domain compositions, we did not detect water, like P. cinnamomi vs. P. sojae had more gene additional orthologs other than one PifHAT3 in the gen- duplication events, whereas, the species commonly attack- ome of T. pseudonana, a diatom (Additional file 10). ing the upper plant part, like P. infestans vs. P. parasitica Phylogenetic analysis revealed that these putative novel experienced more gene losses. Moreover, an interesting HATs derived from different lineage pre-dating the spe- thing is that the abundance of HATs and HDACs in each ciation of these oomycetes (Additional file 10). species doesn’t related to the total gene content of each Moreover, we found some members of HATs and species, but related to the outcome of gene duplication and HDACs with high sequence identity in many faculta- loss events observed in these genes, and the habitat of the tive parasitic species. For example, the SpaHDAC4 species. Since the abundance of duplication events is high (XM_012357993.1) and SpaHDAC5 (XM_012345512.1), in those species of spending most or entire life in water, like and the PrSir2.3 (DS566101) and PrSir2.4 (DS567307) S. parasitica and Py. ultimum, and the abundance of gene have 100 and 100% identity, respectively (Fig. 4). Further- loss is high in the species of living most of or their entire more, aggregation of these members in a short region life on plant, it is likely that possessing multiple copies of on the genome was observed. For example, PyuHAG2 HATs and HDACs is favorable for life in water, but not for (K3XAR0), PyuHAG15 (K3XAR1), and PyuHAG20 living on plants. Therefore, living habitat probably is an im- (K3XAQ0) with 70.5 to 90.6% identity (Additional file 11a) portant driven force shaping the evolution of these genes. Wang et al. BMC Genomics (2016) 17:927 Page 10 of 14

HATs and HDACs 0.16 2 9 44 18,179 Phytophthora infestans 0.02 0 - 12 Duplications 99 0 - 21 Losses 0.16 6 2 56 20,501 Phytophthora parasitica 23 - 60 Gene counts 0.02 0.16 Total gene contents 100 0 6 44 26,584 Phytophthora sojae 0.02 99 0.17 0.06 3 0 53 26,131 Phytophthora cinnamomi 0.03 100 Ecosystem 100 Plants and soil 0.18 5 15 41 15,743 Phytophthora ramorum Plants 0.02 Freshwater 100 0.19 5 5 46 19,805 Phytophthora capsici Lifestyles 0.07 Facultative parasites 0.24 1 18 30 14,567 Hyaloperonospora parasitica Obligate parasites 100 0.11 0.23 100 8 7 48 15,291 Pythium ultimum

0.11 0.24 100 1 21 23 13,804 Albugo laibachii

0.24 12 0 60 20,113 Saprolegnia parasitica

0.29 0 2 24 10,392 Phaeodactylum tricornutum 0.42 100 0.24 0 3 23 11,672 Thalassiosira pseudonana

0.1 Fig. 5 The projected events of gene duplication and loss of HATs and HDACs in ten oomycetes. Neighbor-Joining phylogeny of the analyzed oomycetes was constructed based on proteomes. The numbers of gene duplication and loss events, the total number of HATs and HDACs, as well as the predicted gene content of each species were projected in the boxes on the dash line near each branch. The robustness of the topology was assessed using 100 bootstrap replicates (bold numbers)

Oomycetes have diverse fungus-like eukaryote micro- the genome of the facultative parasites P. ramorum and organisms living in marine, freshwater, and terrestrial P. sojae [44]. Meanwhile, compared to Phytophthora environments [33, 34]. Recent studies revealed that species, reduced RXLR gene number and fewer effector oomycetes evolved from the simple holocarpic marine paralogs were found in H. parasitica [45]. parasites with two trends as, from marine water to fresh water and from marine water to soil and plant. Further, Conserved acetylated sites on histone N-terminal tails of the Saprolegniales (species in fresh water), Pythiales (soil oomycetes born species), Peronosporales, and Albuginales (obligate To investigate conserved acetylated sites, the sequences of parasites on plant) (Fig. 1) are the “crown oomycetes” in histone subunits in these ten oomycetes species were these two evolution trends [70]. So, the high diversity compared with reference species. Based on histone se- and the evidence of gene duplication found in HATs and quence alignments, we found that the histone subunits of HDACs of the facultative parasites in Saprolegniales, oomycetes (H2A, H2B, H3, and H4) have highly con- Pythiales, and Peronosporales exhibits an expansion of served sequences, and many conserved acetylation sites HATs and HDACs in the evolution of oomycetes (Figs. 1 were found on the histone tails of these oomycetes and 5), whereas the significant high frequency of gene (Fig. 6a). The results suggested that 698 out of 1,019 iden- loss outcome the gene duplication events possibly reveal tified lysines on histone subunits could be acetylated, such the contraction of HATs and HDACs in obligated plant as H3K9, H3K14 and H3K56. The Fig. 6b illustrates the parasitic species in the process of adapting to plant. In frequency distribution of amino acids surrounding lysine general, the adequacy and diversity of HATs and HDACs in the histone that could be acetylated. found in oomycetes reflects the evolution of these organisms with the change of habitat. HATs and HDACs of P. infestans are expressed in stages of Previously, several large-scale duplications in genome development, infection and responsive to stress of several plant parasitic Phytophthora [66, 72] and In order to gain insight into the possible role of oomy- constantly gene duplication and loss has been found in cetes HATs and HDACs, we examined the expression of the genomes of pathogenic oomycetes [73]. The rapid predicted HATs and HDACs in ten developmental stages expansion and diversification of many protein families of P. infestans. As similar expression patterns were related to plant infection, such as hydrolases, ABC trans- yielded from two biological replicates (Additional file porters, protein toxins, and a superfamily of proteins 12), the results from the first experiment were used to with similarity to avirulence genes, has been found in draw the heatmap and construct the HCL tree. Our data Wang et al. BMC Genomics (2016) 17:927 Page 11 of 14

K a K K K K K 2 K K K K K K 7 6 3 16 131 K 5 13 14 128 71 17 H2A H2B K K K 19 21 24 23 K K K K 5 K K K 8 4 K K 31 9 K K H3 H4 16 12 14 K 56 18 K 27 K 23 36 37

Fig. 6 Conserved acetylated sites on histone N-termini shared by ten oomycetes species. a Computational predictions of histone acetylation sites on H2A, H2B, H3 and H4. The numbers indicate the positions of amino acids on the histone tails. b Frequency distribution of amino acids surrounding lysines in acetylated and non-acetylated histones. Frequency of amino acids (y axis) spanning positions −6 to 6 (x axis) showed differences in predicted acetylated lysines (698) (left) and non-acetylated lysines (321) (right) in the core histone protein. For sequences of lysines that are located close to each other, only one of them was picked up by analysis. Residues in polar amino acids are indicated in green, and the basic, acidic and hydrophobic amino acids are indicated in blue, red and black, respectively showed that all 33 HATs and 11 HDACs predicted in P. infes- genes, including PifHAG5 (XM_002998432.1) and Pif- tans were exposed at ten developing stages with distinct pat- HAG19 (XM_002903653.1) that hardly up-regulated terns (Fig. 7) that distributed them into six groups (Fig. 7a during asexual and sexual reproduction stage, have acti- and b). For example, genes in group b, including PifHAG12 vated in response to diverse environmental stresses (XM_002900188.1), PifHAG15 (XM_002900191.1),PifHAF1 (Additional file 5) including nutrition starvation and (XM_002908163.1), and PifHAC2 (XM_002997911.1), and heat treatment. PifHDAC5 (XM_002998818.1), were highly expressed at SP Analysis of expression profiles of P. infestans during infec- stage of asexual reproduction and at 4DM and 10DM stage tion (Additional files 5 and 13) also revealed that the of the sexual reproduction of P. infestans, while genes in predicted 33 HATs and 11HDACs genes expressed differently Group e were up-regulated in SP, CY stages as well as in during the infection. PifHAG22 (XM_002907401.1), Pif- sexual reproduction stage. These data also demonstrated that HAM1 (XM_002908075.1),PifHDAC2(XM_002904346.1), the expression pattern of HATs and HDAC in the late and PifHDAC3 (XM_002905236.1) were up-regulated in oospore formation is different than those in the oogonia 2dpi, while 8 another HATs and HDACs including PifHAG21 forming stage but more close to those in the sporangia form- (XM_002909614.1), PifHAT3 (XM_002895006.1), PifHDAC4 ing stage (Fig. 7c, Additional file 12). Strikingly, some genes (XM_002900466.1) and PifSir2.3 (XM_002909246.1) were were specifically highly expressed in certain development down-regulated in 2dpi (Additional files 5 and 13), dem- stage, such as PifHAG15 (XM_002900191.1) and PifHAG18 onstrating that the predicted HATs and HDACs are (XM_002902054.1) were up-regulated more than 100 fold in involved in the interaction between P. infestans and plant. SP stage, while PifHAG20 (XM_002894902.1), PifHAG21 Previously, knowledge of epigenetic mechanisms in (XM_002909614.1) and PifHAG18 (XM_002902054.1) in- oomycetes was not available, except a few indirect evi- creased more than 100 fold at the late oogonia formation dences suggesting that histone acetylation may be in- stage(10DM).TheavailablemicroarraydataandTPMat volved in gene expression [8, 9, 74]. To our knowledge, same development stages in previous study are consist- this is the first study to identify and analyze the evolu- ent with the expression trends found in this study tion and the express pattern of HATs and HDACs in (Additional file 5) [65], except that less genes were oomycetes. detected in previous study due to the unavailable of It is well known that a large genome dataset acceler- genome sequence when those experiments were per- ates gene discovery in an organism. As gene manipula- formed. Analysis of the ESTs data from previous study tion techniques become available in several species of showed that the expression of 12 HATs and 11 HDACs oomycetes, such as P. infestans and P. sojae, dataset Wang et al. BMC Genomics (2016) 17:927 Page 12 of 14

a b c 30 120 ab 22.5 90

a 15 60

b 7.5 30

0 0

200 60 c c d 150 45

100 30

d 50 15 Expression level 0 0

60 60 e e f 45 45

30 30

15 15 f 0 0 SP SP ZO CY ZO CY MY GC MY GC 4D1 4D2 4D1 4D2 4DM 4DM 10DM 14DM 10DM 14DM Fig. 7 Clustering analysis of expression patterns of HATs and HDACs in P. infestans during development stages. The heat map a and the line charts b that generated by the clustering affinity search show the expression data of 33 HATs and 11 HDACs of P. infestans at different biological stages that detected with real-time qPCR. The color bar represents the log2 of expression fold changes, ranging from white (0.0) to blue (12.0). As the sequence identity within each of the two pairs of genes, PifHAG3 and PifHAG24, PifHDAC6 and PifHDAC8, is over than 97%, same primer was used for detecting the expression of each pair of genes. c Hierachical clustering tree of different samples. MY, mycelia; SP, sporangia; ZO, zoospores; CY, cysts; GC, germinated cysts; 4D1(or 2), the 4d old mycelia of A1 (or A2) isolate; respectively; 4DM, 10DM and 14DM, the 4d, 10d, and 14d old mycelia and oospores from junction areas between A1 and A2 isolates resulted from this study will accelerate the investigation HATs and HDACs in oomycetes. In addition, sequence of epigenetic mechanisms in oomycetes. Further study analysis revealed many conserved acetylation sites on of the epigenetic mechanisms in oomycetes will not histones and suggested that 698 out of 1,019 identified only provide us with knowledge to manipulate the lysines could be acetylated. By analyzing the expression biology of these organisms, but will also benefit the data of HATs and HDACs of P. infestans identified in this understanding of epigenetic mechanisms in the many study, we are able to provide an overview of the specific organisms in Stramenopiles. biological stages that these genes involved, which give hints of their functions. Results of this study provide Conclusions useful inputs to help explore the epigenetic mechanisms In this study, we have identified many HATs and HDACs and the relationship between genomes and phenotypes of in oomycetes, along with five histone acetyltransferase oomycetes. families (HAGs, HAFs, HAMs, HACs, and Hat1s), three putative novel HATs, and three classes (I, II and III) of histone deacetylases. We also reveal the structure of these Additional files HATs and HDACs. Phylogenetic analyses have suggested Additional file 1: Gene information for HATs and HDACs of ten oomycetes that most of these HATs and HDACs derived from dis- species and the reference species used in this study. (XLS 301 kb) tinct evolutionary ancestors, and most of them are closely Additional file 2: Orthologous and paralogous groups of HAGs and related to those in marine algae and some other species in HDACs in oomycetes. (XLSX 291 kb) Stramenopiles. Phylogenetic based analysis unveiled the Additional file 3: Primers for real-time qPCR used in this study. complex and distinct patterns of duplications and losses (DOC 156 kb) of HATs and HDACs in oomycetes. This study reveals the Additional file 4: Sequences alignments of the amplified cDNA fragments and the targeted genes. (DOC 116 kb) evolutionary dynamics that shaped the gene content of Wang et al. BMC Genomics (2016) 17:927 Page 13 of 14

Additional file 5: EST, cDNA data and microarray expression values of Availability of data and materials HATs and HDACs of Phytophthora infestans. Data were from NCBI UniGene The datasets supporting the results of this article are available for download via and GEO DataSets. (XLSX 33 kb) the Gene Expression Omnibus repository, GSE14480 and GSE6343. The sequences accession numbers and features are listed in Additional files 1 and 4. Additional file 6: Phylogenetic tree of HAFs in oomycetes. A maximum-likelihood phylogenetic tree was constructed with sequences Authors’ contributions of HAF conserved domains from the species described in Fig. 2. Each L-YG and X-WW designed the experiments. X-WW, MH and KS performed domain was highlighted by one color and bootstrap values (≥50%) are the experiments and analyzed the data. X-WW and L-YG were involved in shown near the tree nodes. (PDF 194 kb) writing the manuscript. All authors reviewed the manuscript. All authors read Additional file 7: Phylogenetic tree of HACs in oomycetes. A maximum- and approved the final manuscript. likelihood phylogenetic tree was constructed with sequences of HAC conserved domains from the species described in Fig. 2. All the predicted Competing interests HACs of oomycetes were attributed to four clades according to the The authors declare that they have no competing interests. combination of conserved domains. These domains were highlighted by different colors and bootstrap values (≥50%) are shown near the tree Consent for publication nodes. (PDF 423 kb) Not applicable. Additional file 8: Phylogenetic tree of HAMs in oomycetes. A maximum-likelihood phylogenetic tree was constructed with sequences Received: 21 July 2016 Accepted: 9 November 2016 of HAM conserved domains from the species described in Fig. 2. Different domains were highlighted by different colors and bootstrap values ≥ ( 50%) are shown near the tree nodes. (PDF 303 kb) References Additional file 9: Phylogenetic tree of Hat1s in oomycetes. A 1. Hattori N, Ushijima T. Compendium of aberrant DNA methylation and maximum-likelihood phylogenetic tree was constructed with sequences histone modifications in cancers. Biochem Bioph Res Co. 2014;455:3–9. of Hat1 conserved domains from the species described in Fig. 2. Each 2. Ehrlich M, Gama-Sosa MA, Huang LH, Midgett RM, Kuo KC, McCune RA, Oomycetes species contained one Hat1 and they were attributed to 1 et al. Amount and distribution of 5-methycytosine in human DNA from clade. Each domain was highlighted by one color and bootstrap values different types of tissues or cells. Nucleic Acids Res. 1982;10(8):2709–21. (≥50%) are shown near the tree nodes. (PDF 201 kb) 3. Lister R, Pelizzola M, Dowen RH, Hawkins RD, Hon G, Tonti-Filippini J, et al. Additional file 10: Phylogenetic tree of putative novel HATs in Human DNA methylomes at base resolution show widespread epigenomic – oomycetes. A maximum-likelihood phylogenetic tree was constructed differences. Nature. 2009;462(7271):315 22. – with sequences of the conserved domain AT-1 found in the species 4. Bestor TH. Cloning of a mammalian DNA methltransferase. Gene. 1988;74:9 12. described in Fig. 2. Different domains were highlighted by different colors 5. Okano M, Xie S, Li E. Cloning and characterization of a family of novel – and bootstrap values (≥50%) are shown near the tree nodes. (PDF 189 kb) mammalian DNA (cytosine-5) methyltransferases. Nat Genet. 1998;19:219 20. 6. Yoder JA, Bestor TH. A candidate mammalian DNA methytransferase related Additional file 11: Sequence alignment of some Hpa2s in oomycetes. to pmt1 of fission yeast. Hum Mol Genet. 1998;7:279–84. ≥ Bootstrap values ( 30%) and sequence identity are shown near the tree 7. Jin B, Li Y, Robertson KD. DNA methylation: superior or subordinate in the nodes. a Members of Hpa2 in the same species with high sequence Epigenetic hierarchy? Genes Cancer. 2011;2:607–17. identity aggregated in the same scaffold. b Members of Hpa2 in the 8. Judelson HS, Tani S. Transgene-induced silencing of the zoosporogenesis- same species with various sequence identities clustered in the same specific NIFC gene cluster of Phytophthora infestans involves chromatin scaffold. (PDF 421 kb) alterations. Eukaryot Cell. 2007;6:1200–9. Additional file 12: Gene expression data of real-time qPCR in this study. 9. van West P, Shepherd SJ, Walker CA, Li S, Appiah AA, Grenville-Briggs LJ, (XLS 313 kb) et al. Internuclear gene silencing in Phytophthora infestans is established – Additional file 13: Expression profiles of HATs and HDACs in P. infestans through chromatin remodeling. Microbiol. 2008;154:1482 90. at various stages during infection. The heat map generated by microarray 10. LugerK,MäderAW,RichimondRK,SargentDF,RichmondTJ.Crystal expression values of GSE14480 retrieved from GEO DataSets. The color structure of the nucleosome core particle at 2.8 Å resolution. Nature. 1997;389:251–60. bar represents the log2 of expression values, and the highest and lowest log2 signal value is 8.62 and 12.97, respectively. (PDF 62 kb) 11. Ma X, Lv S, Zhang C, Yang C. Histone deacetylases and their functions in plants. Plant Cell Rep. 2013;32:465–78. 12. Phillips DM. The presence of acetyl groups of histones. Biochem J. Abbreviations 1963;87:258–63. acetyl CoA: Acetyl coenzyme A; DNMTs: DNA methyltransferases; 13. Allfrey VG, Faulkner R, Mirsky AE. Acetylation and methylation of histones Elp3: Elongator proteins 3; Gcn5: General control nonderepressible 5; and their possible role in the regulation of RNA synthesis. Proc Natl Acad HATs: Histone acetyltransferase; HDACs: Histone deacetylase; Sci U S A. 1964;51:786–94. Hpa2: Heparanase 2; p300/CBP: CREB-binding protein; PTMs: Post- 14. Brownell JE, Allis CD. Special HATs for special occasions: linking histone translational modifications; RPD3: Reduced potassium dependency acetylation to chromatin assembly and gene activation. Curr Opin Genet 3; Sir2: Silent information regulator 2; TAFII250: TBP-associated Dev. 1996;6:176–84. factor subunits of TFIID 15. Pflum MK, Tong JK, Lane WS, Schreiber SL. Histone deacetylase 1 phosphorylation promotes enzymatic activity and complex formation. J Biol Acknowledgements Chem. 2001;276:47733–41. We thank Drs. Qun He and Yuan-biao Zhao (State Key Laboratory of 16. Bertos NR, Wang AH, Yang XJ. Class II histone deacetylases: structure, Agro-biotechnology and MOA Key Laboratory of Soil Microbiology, CAU) function, and regulation. Biochem Cell Biol. 2001;79:243–52. and Dr. Boming Wu (Department of Plant Pathology, China Agricultural 17. Thiagalingam S, Cheng KH, Lee HJ, Mineva N, Thiagalingam A, Ponte JF. University) for reviewing this manuscript, Dr. Timothy Y. James (University Histone deacetylases: unique players in shaping the epigenetic histone of Michigan), Dr. Suomeng Dong (Nanjing Agricultural University), code. Ann N Y Acad Sci. 2003;983:84–100. Dr. Yongjie Zhang (Shanxi University) for valuable suggestion and 18. Hong L, Schroth GP, Matthews HR, Yau P, Bradbury EM. Studies of the DNA Dr. Horward S Judelson (University of California, Riverside) for providing finding properties of histone H4 amino terminus: thermal denaturation the gene information of microarray expression. studies reveal that acetylation markedly reduces the binding constant of the H4 “tail” to DNA. J Biol Chem. 1993;268:305–14. Funding 19. Mukherjee K, Fischer R, Vilcinskas A. Histone acetylation mediates epigenetic This research was funded in part by the National Natural Science regulation of transcriptional reprogramming in insects during Foundation of China (30270862) and the Chinese Universities Scientific metamorphosis, wounding and infection. Front Zool. 2012;9:25. doi:10.1186/ Fund (2015NX005). 1742-9994-9-25. Wang et al. BMC Genomics (2016) 17:927 Page 14 of 14

20. Pandey R, Müller A, Napoli CA, Selinger DA, Pikaard CS, Richards EJ, et al. Analysis 49. de Bruijn I, Belmonte R, Anderson VL, Saraiva M, Wang T, van West P, et al. of histone acetyltransferase and histone deacetylase families of Arabidopsis Immune gene expression in trout cell lines infected with the fish pathogenic thaliana suggests functional diversification of chromatin modification among oomycete Saprolegnia parasitica. Dev Comp Immunol. 2012;38(1):44–54. multicellular eukaryotes. Nucleic Acids Res. 2002;30:5036–55. 50. Lévesque CA, Brouwer H, Cano L, Hamilton JP, Holt C, Huitema E, et al. 21. Shahbazian MD, Grunstein M. Functions of site-specific histone acetylation Genome sequence of the necrotrophic plant pathogen Pythium ultimum and deacetylation. Annu Rev Biochem. 2007;76:75–100. reveals original pathogenicity mechanisms and effector repertoire. Genome 22. Yang XJ, Seto E. Lysine acetylation: codified crosstalk with other Biol. 2010;11(7):R73. doi:10.1186/gb-2010-11-7-r73. posttranslational modifications. Mol Cell. 2008;31:449–61. 51. Letunic I, Doerks T, Bork P. SMART: recent updates, new developments and 23. Grant PA, Berger SL. Histone acetyltransferase complexes. Cell Dev Biol. status in 2015. Nucleic Acids Res. 2015;43:D257–60. doi:10.1093/nar/gku949. 1999;10:169–77. 52. Marchler-Bauer A, Lu S, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese- 24. Alonso VL, Serra EC. Lysine acetylation: Elucidating the components of an Scott C, et al. CDD: a Conserved Domain Database for the functional emerging global signaling pathway in Trypanosomes. J Biomed Biotech. annotation of proteins. Nucleic Acids Res. 2011;39:225–9. 2012;2012:452934. doi:10.1155/2012/452934. 53. Sigrist CJ, de Castro E, Cerutti L, Cuche BA, Hulo N, Bridge A, et al. New and 25. Marmorstein R, Roth SY. Histone acetyltransferases: function, structure, and continuing developments at PROSITE. Nucleic Acids Res. 2013;41:344–7. catalysis. Curr Opin Genet Dev. 2001;11:155–61. 54. Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY, Eddy SR, et al. 26. Kleff S, Andrulis ED, Anderson CW, Sternglanz R. Identification of a gene Pfam: the protein families database. Nucleic Acids Res. 2014;42:222–30. encoding a yeast histone H4 acetyltransferase. J Biol Chem. 1995;270:24674–7. 55. Buchan DW, Minneci F, Nugent TC, Bryson K, Jones DT. Scalable web services for 27. Roth SY, Denu JM, Allis CD. Histone acetyltransferase. Annu Rev Biochem. the PSIPRED Protein Analysis Workbench. Nucleic Acids Res. 2013;41:W349–57. 2001;70:81–120. 56. Chou PY, Fasman GD. Conformational parameters for amino acids in helical, 28. de Ruijter AJ, van Gennip AH, Caron HN, Kemp S, van Kuilenburg AB. beta-sheet, and random coil regions calculated from proteins. Histone deacetylases (HDACs): characterization of the classical HDAC family. Biogeosciences. 1974;13:211–22. Biochem. 2003;370:737–49. 57. Crooks GE, Hon G, Chandonia JM, Brenner SE. WebLogo: A sequence logo 29. Afshar G, Murnane JP. Characterization of a human gene with sequence generator. Genome Res. 2004;14(6):1188–90. homology to Saccharomyces cerevisiae SIR2. Gene. 1999;234:161–8. 58. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, 30. Frye RA. Phylogenetic classification of prokaryotic and eukaryotic Sir2-like et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007;23(21):2947–8. proteins. Biochem Biophys Res Commun. 2000;273:793–8. 59. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: 31. Marmorstein R. Structure and function of histone acetyltransferases. Cell Mol molecular evolutionary genetics analysis using maximum likelihood, Life Sci. 2001;58:693–703. evolutionary distance, and maximum parsimony methods. Mol Biol Evol. – 32. Basu A, Rose KL, Zhang J, Bravis RC, Ueberheide B, Garcia BA, et al. 2011;28(10):2731 9. Proteome-wide prediction of acetylation substrates. Proc Natl Acad Sci U S 60. Qi J, Luo H, Hao B. CVTree: a phylogenetic tree reconstruction tool based – A. 2009;106(33):13785–90. on whole genomes. Nucleic Acids Res. 2004;32:W45 7. 33. Sparrow FK. Aquatic Phycomycetes 2nd revised edition. Michigan: University 61. Chen K, Durand D, Farach-Colton M. NOTUNG: a program for dating gene – of Michigan Press, Ann Arbor; 1960. 1187. duplications and optimizing gene family trees. J Comput Biol. 2000;7:429 47. 34. Karling JS. Predominantly holocarpic and eucarpic simple biflagellate 62. Durand D, Halldórsson BV, Vernot B. A hybrid micro-macroevolutionary – phycomycetes. 2nd edition. Germany: J. Cramer; 1981. 252. approach to gene tree reconstruction. J Comput Biol. 2006;13:320 35. 35. Rizzo DM, Garbelotto M, Hansen EM. Phytophthora ramorum: integrative 63. Basu A, Rose KL, Zhang J, Beavis RC, Ueberheide B, Garcia BA, et al. research and management of an emerging pathogen in California and Proteome-wide prediction of acetylation substrates. Proc Natl Acad Sci USA. – Oregon forests. Annu Rev Phytopathol. 2005;43:309–35. 2009;106(33):13785 90. 36. Harham AR. Pathogen profile: Phytophthora cinnamomi. Mol Plant Path. 64. Guo LY, Zhu XQ, Hu CH, Ristaino JB. Genetic structure of Phytophthora 2005;6:598–604. infestans populations in China indicates multiple migration events. – 37. Torto-Alalibo T, Tian M, Gajendran K, Waugh ME, van West P, Kamoun S. Phytopathol. 2010;100(10):997 1006. Expressed sequence tags from the oomycete fish pathogen Saprolegnia 65. Prakob W, Judelson HS. Gene expression during oosporogenesis in heterothallic – parasitica reveal putative virulence factors. BMC Microbiol. 2005;5:46. and homothallic Phytophthora. Fungal Genet Biol. 2007;44(8):726 39. 66. Haas BJ, Kamoun S, Zody MC, Jiang RH, Handsaker RE, Cano LM, et al. 38. Richards TA, Dacks JB, Jenkinson JM, Thornton CR, Talbot NJ. Evolution of Genome sequence and analysis of the Irish potato famine pathogen filamentous pathogens: gene exchange across eukaryote kingdoms. Curr Phytophthora infestans. Nature. 2009;461(7262):393–8. Biol. 2006;16(18):1857–64. 67. Dhalluin C, Carlson JE, Zeng L, He C, Aggarwal AK, Zhou MM. Structure 39. Baldauf SL. The deep roots of eukaryotes. Science. 2003;300:1703–6. and ligand of a histone acetyltransferase bromodomain. Nature. 1999; 40. Sogin ML, Silberman JD. Evolution of the protists and protistan parasites 399(6735):491–6. from the perspective of molecular systematics. Int J Parasitol. 1998;28:11–20. 68. Tang Y, Holbert MA, Wurtele H, Meeth K, Rocha W, Gharib M, et al. Fungal 41. Baldauf SL, Roger AJ, Wenk-Siefert I, Doolittle WF. A kingdom-level Rtt109 histone acetyltransferase is an unexpected structural homolog of phylogeny of eukaryotes based on combined protein data. Science. metazoan p300/CBP. Nat Struct Mol Biol. 2008;15(7):738–45. 2000;290(5493):972–7. 69. Mersfelder EL, Parthun MR. Involvement of Hat1p (Kat1p) catalytic activity 42. Yoon HS, Hackett JD, Pinto G, Bhattacharya D. The single, ancient origin of and subcellular localization in telomeric silencing. J Biol Chem. 2008;283: chromist plastids. Proc Natl Acad Sci U S A. 2002;99(24):15507–12. 29060–8. 43. Adl SM, Simpson AG, Farmer MA, Andersen RA, Anderson OR, Barta JR, et al. 70. Beakes GW, Glocking SL, Sekimoto S. The evolutionary phylogeny of The new higher level classification of eukaryotes with emphasis on the oomycete “fungi”. Protoplasma. 2012;249:3–19. taxonomy of protists. J Eukaryot Microbiol. 2005;52(5):399–451. 71. Kroon LP, Brouwer H, de Cock AW, Govers F. The genus Phytophthora anno 44. Tyler BM, Tripathy S, Zhang X, Dehal P, Jiang RH, Aerts A, et al. Phytophthora 2012. Phytopathol. 2012;102(4):348–64. genome sequences uncover evolutionary origins and mechanisms of 72. Martens C, Van de Peer Y. The hidden duplication past of the plant pathogenesis. Science. 2006;313(5791):1261–6. pathogen Phytophthora and its consequences for infection. BMC 45. Coates ME, Beynon JL. Hyaloperonospora arabidopsidis as a pathogen model. Genomics. 2010;11:353. – Annu Rev Phytopathol. 2010;48:329 45. 73. Seidl MF, Van den Ackerveken G, Govers F, Snel B. Reconstruction of 46. Hausbeck MK, Lamour KH. Phytophthora capsici on vegetable crops: oomycete genome evolution identifies differences in evolutionary research progress and management challenges. Plant Dis. 2004;88: trajectories leading to present-day large gene families. Genome Biol Evol. – 1292 303. 2012;4(3):199–211. doi:10.1093/gbe/evs003. 47. Meng Y, Zhang Q, Ding W, Shan W. Phytophthora parasitica: a model 74. Vetukuri RR, Avrova AO, Grenville-Briggs LJ, Van West P, Söderbom F, – oomycete plant pathogen. Mycol. 2014;5(2):43 51. Savenkov EI, et al. Evidence for involvement of Dicer-like, Argonaute and 48. KemenE,GardinerA,Schultz-LarsenT,KemenAC,BalmuthAL, histone deacetylase proteins in gene silencing in Phytophthora infestans. Robert-Seilaniantz A, et al. Gene gain and loss during evolution of Mol Plant Pathol. 2011;12(8):772–85. obligate parasitism in the white rust pathogen of Arabidopsis thaliana. PLoS Biol. 2011;9(7):e1001094.