QlAN Kun, Ll Deng-hui, LlN Run-mao, SHl Qian-qian MAO Zhen-chuan YANG Yu-hong FENG Dong-xin XlE Bing-yan

1 Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China

2 College of Life Sciences, Beijing Normal University, Beijing 100875, P.R.China

3 Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, Beijing 100081, P.R.China

1. lntroduction

Phytophthora, a genus well-known worldwide, includes several plant pathogens used as model organisms,includingPhytophthora infestans, the microorganism primarily responsible for potato late blight (Judelson and Blanco 2005). Over 100 species ofPhytophthorahave been recognized (Kroonet al. 2011). The expansion of global trade in agricultural products has facilitated the spread of many pathogenic species, together with their hosts, beyond their regions of origins. As a result,epidemics of plant diseases are frequently reported worldwide; for example, sudden oak death in western North America and Europe caused byPhytophthora ramorum(Hansen 2008).Phytophthorapathogens are notorious for the damage they cause to plants, which results in major economic losses; for example, the crop destructive pathogenP.infestans, dubbed the cancer of the potato(Large 1940) after its role in the Irish potato famine, causes about $45.5 billion worth of losses of potato crops annually(Ristaino 2002; Haverkortet al. 2008).

The publications of the whole genome sequences ofP.ramorum,Phytophthora sojae(Tyleret al. 2006),P.infestans(Haaset al. 2009),Phytophthora capsici(Lamouret al. 2012),Phytophthora lateralis(Quinnet al.2013) andPhytophthora nicotianae(Liuet al. 2016) have led to a new era of investigation of genomic information and gene regulation inPhytophthoraspecies (Table 1). Previous analysis of these genomes has identified virulence genes involved in pathogenesis, including cytoplasmic effectors such as Arg-Xaa-Leu-Arg (RXLR) and Crinkler, and other potential infection-related genes that may play critical roles in pathogenicity, including proteinase inhibitors, protein toxins, secondary metabolites, ATP-binding cassette (ABC)transporters, and hydrolases (Kamoun 2006; Tyleret al.2006; Haaset al. 2009). Some secreted pathogenic factors have been designated as Carbohydrate-Active enZymes(CAZymes) and classified in the Carbohydrate-Active Enzymes (CAZy) database (Cantarelet al. 2009; Lombardet al. 2014), which contains the six major classes, glycoside hydrolases (GHs), glycosyltransferases, polysaccharide lyases (PLs), CEs, enzymes with auxiliary activities, and carbohydrate-binding modules. CAZymes have multiple roles in biology; for example, GHs, PLs and CEs are likely to act as cell wall degrading enzymes in fungi (Ospina-Giraldoet al. 2003; Ospina-Giraldoet al. 2010a; Aspeborget al. 2012). In addition, some CAZymes are involved in both xylogenesis and lignocellulosic biomass production in plants and bacteria (Menget al. 2015; Pinardet al. 2015).Hence, CAZymes are significant factors in pathogenic mechanisms and pathogen-host interactions. For hosts,to protect plant cell walls against invading microorganisms,some plant polysaccharides have been evolved to alter their chemical structure by acetylation of glycosyl residues(Biely 2012). However, microbes and host plants coevolution and invading microorganisms have developed weapons, consisting of CEs and their co-operative GHs,to overcome the acetyl groups (Biely 2012). The CE superfamily comprises 16 sub-families, most of which bind polysaccharide substrates and catalyze the hydrolysis of esters (Ospina-Giraldoet al. 2010b).

例如，在教學(xué)北師大版三年級(jí)數(shù)學(xué)“兩位數(shù)乘兩位數(shù)”時(shí)，我先讓學(xué)生對整十的兩位數(shù)進(jìn)行計(jì)算，然后讓學(xué)生在課后探究學(xué)校馬上進(jìn)行的“隊(duì)列表演”這一活動(dòng)，讓學(xué)生再具體的生活情境中探究數(shù)學(xué)問題，讓學(xué)生用點(diǎn)子圖或者列表的形式探究兩位數(shù)乘兩位數(shù)的橫式筆算的過程與方法。學(xué)生通過課后探究，把課堂的數(shù)學(xué)學(xué)習(xí)經(jīng)驗(yàn)和生活中實(shí)踐經(jīng)驗(yàn)相互融合，產(chǎn)生了新的思維和學(xué)習(xí)方法。學(xué)生對數(shù)學(xué)的學(xué)習(xí)逐漸產(chǎn)生真實(shí)感，學(xué)到的數(shù)學(xué)知識(shí)也就有生活價(jià)值和意義。

Cellulose, hemicellulose, and pectin are abundant components of plant cell walls (Somervilleet al. 2004; Glasset al. 2013). Hemicelluloses include xyloglucans, xylans,mannans, glucomannans, and (1,3;1,4)-β-glucans (Scheller and Ulvskov 2010), whereas pectins have four major forms,homogalacturonan, xylogalacturonan, rhamnogalacturonan I,and rhamnogalacturonan II (Mohnen 2008; Harholtet al.2010). Many CE enzymes are involved in xylan and pectin degradation (Glasset al. 2013), including CE1 (acetyl xylan esterases; ferulic acid esterases), CE2–CE7 (acetylxylan esterases), CE8 (homogalacturonan methylesterases), CE12(homogalacturonan acetylesterases; rhamnogalacturonan I acetylesterases) and CE13 (homogalacturonan acetylesterases), which has stimulated research on CE enzymes in plant pathogens, including studies aimed at gene discovery, functional evaluation, and evolutionary analyses.Pectin acetyl esterases remove the acetyl group from homogalacturonan and rhamnogalacturonan I (Sénéchalet al. 2014).

Table 1 Reported genomes of sequenced oomycete pathogens

In recent years, several powerful tools have been developed to investigate CAZymes, including the CAZy database (Lombardet al. 2014), the CAZymes Analysis Toolkit (CAT) web server (Parket al. 2010), and the default parameters (dbCAN) web server (Yinet al. 2012).Meanwhile, the Pfam database (Finnet al. 2014) and the NCBI Conserved Domain Database (CDD) (Marchler-Baueret al. 2015) can be used to identify CE functional domains.With these publically available genomic resources and analysis tools, some methods have been developed with the aim of discovering CE genes inPhytophthora(Ospina-Giraldoet al. 2010a, b; Zerilloet al. 2013; Brouweret al.2014). However, the results of these methods have been reported as highly variable (Appendix A), indicating the significance of rediscovery of CEs and providing credible annotations for these genes. The identification of CE genes inPhytophthorais not only significant for investigation of their roles in pathogenesis, but also essential for exploration of their evolutionary relationships.

In our study, we propose a pipeline based on Pfam annotations and information from related orthologous group genes, as well as their phylogenetic relationships,to identify CE genes. The application of this method inPhytophthoraled to the discovery of many new putative CEs and prevented proteases in the prediction. We performed comparative analysis using the identified sequences,which revealed the presence of more CE8, CE12, and CE13 genes inPhytophthorathan in two other genera(HyaloperonosporaandPythium). Furthermore, the CE13 gene sequences were used to investigate their phylogenetic relationships in oomycetes during evolution. To explore the associations ofPhytophthoraCE13 with other genes,we used the interolog method (Lehner and Fraser 2004)to investigate associated paired-genes based on reported associated genes fromP.infestans(Seidlet al. 2013) that integrated reported data about interactions, co-expression,and co-occurrence of proteins. Our results show that the combination of orthologous group analysis with conserved functional domain annotation facilitates the discovery of putative CE genes, and the comparative analysis ofPhytophthoraCEs may provide clues to understanding the evolution of these genes.

2. Materials and methods

2.1. Annotation of carbohydrate esterase genes

Based on the collected CE gene sequences in the CAZy database (Parket al. 2010), we identified 11 domain classes (14 Pfam families) representing major functions of CE genes, including polysaccharide deacetylase(PF01522, PF04748), esterase (PF00756), esterase polyβ-hydroxybutyrate (PHB) depolymerase (PF10503), UDP-3-O-acyl N-acetylglycosamine deacetylase (PF03331),GlcNAc-PI de-N-acetylase (PF02585), GDSL-like lipase/acylhydrolase (PF00657, PF13472, PF14606), cutinase(PF01083), carbohydrate esterase (PF03629), acetyl xylan esterase (PF05448), pectinesterase (PF01095), and pectinacetylesterase (PF03283). To identify candidate CE genes, we annotated domains by searching gene sequences from the 16 genomes against sequences in the Pfam database (Finnet al. 2014) using HMMER (Wheeler and Eddy 2013) with E-value cutoff of 1e-5. Then we used OrthoMCL (Liet al. 2003) to identify orthologous groups of candidate CEs from Pfam annotations. In the analysis, we used phylogenetic analysis to distinguish genes from CE2,CE3 and CE12 (Fig. 1-A), for all of them were annotated by GDSL-like lipase/acylhydrolase domains and it was difficult to identify sub-families for these genes based on sequences.And six orthologous groups that contained some genes annotated by CE1 or CE2, and some were also predicted by MEROPS analysis (Rawlingset al. 2016), were removed.Finally, we identified 445 putative CEs (Table 3) belonging to 97 orthologous groups (Appendix E) and compared the results with those from CAT and dbCAN annotations (Fig. 1;Appendices B–H).

To identify secreted proteins, we used the similar methods as in previous studies (Maet al. 2010; Zhenget al. 2013;Wanget al. 2016), which considered proteins to be secreted proteins if they had signal peptides detected by at least two methods among SignalP version 4.0 (Petersenet al. 2011),TargetP version 1.1 (Emanuelssonet al. 2000), Phobious version 1.01 (K?llet al. 2004) and Predisi (Hilleret al. 2004),and did not have transmembrane sequences determined by at least one of the methods among SignalP, Phobious and TMHMM version 2.0c (Kroghet al. 2001).

2.2. Phylogenetic analysis

We performed CAT and dbCAN analyses to identify putative CE genes in 16 oomycete genomes. The CAT method is based on sequence similarity and involves searching protein sequences against data in the CAZy database (Parket al. 2010), and may also incorporate evidence from the Pfam domain database. On the dbCAN web server, hidden Markov models are implemented (Yinet al. 2012) to search for CE family genes. The results demonstrated that both methods identified almost the same CE2, CE4, CE5, and CE8 genes; however, more CE11 genes were predicted by CAT than dbCAN and, conversely, more CE1, CE10, CE12, and CE13 genes were found by dbCAN than CAT (Table 2), suggesting that more attention should be paid to discover CE1, CE10, CE11, CE12 and CE13 genes. Some of them may be proteases by considering their annotations. We performed BLAST analysis on MEROPS database web server (Rawlingset al. 2016) to determine the contaminated protease genes. This analysis supported the high accuracy of gene discovery in CE3, CE4, CE8, CE11,CE12, CE13, CE14 and CE15 families by the CAT and dbCAN methods, as no proteases were identified in these eight sub-families; meanwhile, 635 putative proteases were detected that were mainly distributed in CE1, CE2, CE9 and CE10 sub-families (Table 2; Appendices B–D), suggesting more strict thresholds should be used or further functional annotations were required to identify these genes.

In our CE discovered pipeline, proteases were excluded(Appendix F; Fig. 1-A and D), and genes newly annotated only by conserved functional domains and evolutionary relationships derived from orthologous group information were determined in 16 oomycete genomes (Appendix E;Fig. 1), improving previous CE gene discovery (Fig. 1-C,H, I, M, N, and O). A comparison of putative CE discovery among three methods showed that more CEs were identified by PLdcg than by CAT, and higher accuracy was obtained by PLdcg (72.48%) than by dbCAN (46.68%) (Fig. 1-B–D;Appendices B–F). Although more CEs were identified by dbCAN, which was mainly due to more of 110 CE1 predicted by CE1 (Fig. 1-E), more CE4, CE5, CE12, CE13, and CE14 were identified by PLdcg (Fig. 1-H, I, and M–O). For 13 genes predicted as CE3 by dbCAN, their phylogenetic relationships by GDSL-like lipase/acylhydrolase domains showed that they were clustered into two clades and were closely related to CE12 genes (Appendix H). Among these CE genes, a total of 251 secreted proteins were identified(Appendix G). The analysis suggested that PLdcg that integrated conserved functional domains, orthologous and phylogenetic information is good at discovering CE genes.Genes in orthologous groups are generally considered to share common functional characteristics, arising as the result of speciation events (Koonin 2005). In addition to suggesting the evolutionary relationships of CEs across species, the groups imply the occurrence of gene duplication and gene loss events in these species, which can be integrated with phylogeny to further elucidate the hierarchical relationships among CE genes (Fig. 3). Some genes were assigned to groups consisting of a single gene by OrthoMCL analysis(Liet al. 2003), because they did not meet the algorithm requirements (e.g., alignment coverage) for clustering with other genes. Meanwhile, our results may suggest that a large proportion of sequences were altered for they have multiple copies. In the CE13 phylogeny that included three clades (F, G, and H), the clade H contained genes fromPhytophthoraandPythiumstrains; however, the clade F and clade G contained five units of gene clusters forming byPhytophthoragenes (such as 7 genes fromP.nicotianaerace 0; Fig. 3). Among these five units, noPythiumgene is found, which might support the significant abundance of CE13 genes inPhytophthoracompared withPythiumstrains. ThePythiumstrains are known to infect a broad range of hosts (Lévesqueet al. 2010; Adhikariet al. 2013).The ability of pathogens to infect a wide range of hosts may be a requirement for adaption to varied and complex environmental pressures. The different CE13 gene clades in Fig. 3 indicate that their multiple copies may be related to the host ranges of pathogens, albeit more evidence is required to support this hypothesis. Moreover, the presence and absence of CE13-associated proteins (Seidlet al. 2013)suggest evolutionary diversity among these genes (Fig. 4).The CE13 associated partners (such as PITG_15980,a putative glycoside hydrolase) may act together with pectinacetylesterase to achieve plant penetration. Although the mechanisms of action of pectinacetylesterases and their associations inPhytophthoraremain poorly understood, the loss or gain of pectinacetylesterase-associated proteins in different pathogens identified by the interolog method may reflect pathogen adaptations to different living environments and hosts. Our results provide data useful for future experiment to investigate CE functions inPhytophthora,and suggest an efficient method to discover putative CE genes in other species.

2.3. Functional associations of genes

Known protein associations inP.infestanswere collated from a previous study (Seidlet al. 2013). To identify homologous associations in otherPhytophthoraspecies,the homologous interaction discovery method used in previous studies (Lehner and Fraser 2004; Leiet al. 2014)was performed using the InParanoid algorithm version 4.1(?stlundet al. 2010) with an interaction score cutoff of 0.5.Gene ontology (GO) annotation of associated proteins was performed using BLAST2GO (Conesa and G?tz 2008) with BLASTX E-value cutoff of 1e-5.

3. Results

3.1. Detection of CE genes by CAT and dbCAN analysis

The reported genome sequences of oomycetes containing gene sets are a significant data resource for comparative and evolutionary analyses. Based on 16 genome sequences from the three genera,Phytophthora,HyaloperonosporaandPythium(Table 1), several CE genes potentially involved in pathogenic processes have been predicted by various approaches (Ospina-Giraldoet al. 2010a, b; Zerilloet al.2013; Brouweret al. 2014), including retrieving genes with reported relevant functions (Lombardet al. 2014),CAT annotation (Parket al. 2010), dbCAN annotation (Yinet al. 2012), and homology-based discovery. However,different results were obtained in these studies (Appendix A), indicating that prediction results varied with thresholds in these methods and credible discoveries are restricted to certain thresholds.

The 69 CE13 genes formed three major clusters (clades F, G, and H) in the generated NJ tree (Fig. 3). All genes in clade F forming into three units came fromPhytophthoraspecies, each unit representing a gene topology similar to the species tree in Fig. 2; and most genes (except two genes fromPythium vexans) in clade G were found inPhytophthoraspecies, forming into two units and each displaying a species tree like gene topology. In clade H,a gene unit presenting a species tree like topology was identified as well, however, most genes in this clade came fromPythiumspecies. The phylogeny analysis showed multiple copies of CE13 genes fromPhytophthoraspecies.In addition, the interesting phenomenon of functional domain duplication was identified inPythiumCE13 genes,including two pectinacetylesterase domains in the N-and C-terminal regions of both PAG1_06709 (Pythiumaphanidermatum) and PVE_02897 (P.vexans) proteins(Fig. 3). The abundance of CE13 genes inPhytophthora(46 genes, 66.68%; including 39 secreted proteins), which suggests that these genes may be required for infection by these pathogens, could be explained by the repetitive gene units each displaying species tree like topology, albeit the mechanisms remain poorly understood.

Phylogenetic trees of 16 genomes were constructed based on 111 single-copy genes using Tree-puzzle (Schmidtet al. 2002) with the Dayhoff model (Dayhoffet al. 1978),and multiple sequence alignments were analyzed using MUSCLE version 3.8.31 (Edgar 2004) and neighborjoining (NJ) phylogenies of CE13 genes were generated using p-distance model (bootstrap 1 000) implemented in TreeBeST (http://treesoft.sourceforge.net/treebest.shtml).

3.2. A pipeline of discovering CE genes (PLdcg)

From the dbCAN results, many CE genes are discovered to share conserved domain sequences. For those genes that evolved from the same ancestor and display highly conserved sequences in regions other than the functional domains (where various mutations exist), orthologous group detection methods are helpful for the prediction, as they take into consideration full-length sequences. Therefore,we proposed a pipeline (PLdcg) to discover putative CEs based on functional domain annotations and orthologous gene relationships (Fig. 1-A).

We collected gene sequences from eightPhytophthoragenomes and eight additional genomes from the generaHyaloperonosporaandPythiumfor comparative analysis(Table 1). The amino acid sequences of 16 species were submitted to the CAT (Parket al. 2010; Lombardet al.2014) and dbCAN (Yinet al. 2012) web servers to identify CAZymes genes, using an E-value cutoff of 1e-50 and default parameters, respectively. The Pfam database (Finnet al. 2014) and the CDD (Marchler-Baueret al. 2015)were used to identify domains encoded by CE genes. For Pfam domain discovery, HMMER version 3.1b1 (Wheeler and Eddy 2013) was used with an E-value cutoff of 1e-5.To identify orthologous groups of candidate CE genes annotated by Pfam domains, we performed OrthoMCL (Liet al. 2003) analysis using BLASTP (cutoff E-value of 1e-5,sequence alignment ≥50%) (Altschulet al. 1997). For the analysis, we provided the program ‘PLdcg’ (a pipeline to discover CE genes) on the Github website (https://github.com/Qian-Kun/PLdcg), which could be used to discover putative CEs.

3.3. Distribution of predicted CE genes in the Phytophthora genus

The 445 putative CE genes predicted by PLdcg were distributed in 16 genomes, with numbers of genes ranging from 8 inPythium ultimumvar.sporangiiferumBR650 to 68 inP.sojaeP6497 (Fig. 2; Table 3). There were significantly more CE genes inPhytophthoraspp. than in other genera(HyaloperonosporaandPythium) (Wilcoxon rank sum tests,P＜0.01). All predicted CEs could be classified into 11 sub-families, and the phenomenon of excess genes inPhytophthoracompared with other genera was also observed for three sub-families (CE8, pectinesterase; CE12, GDSL-like lipase/acylhydrolase; and CE13, pectinacetylesterase)(Wilcoxon rank sum tests,P＜0.05). From the phylogenetic analysis, there was a clear evidence of a separation event betweenPhytophthoraandHyaloperonospora, withPythium vexansacting as an outgroup (Fig. 3). The higher number of CEs inPhytophthoracompared withHyaloperonosporaandPythiummay indicate distinct evolutionary histories for somePhytophthoraCEs. Considering the distribution of sub-family members inPhytophthora, CE8 (pectinesterase)genes were abundant, with more than five genes in each of the eight genomes (Table 3). The majority ofPhytophthoragenomes also encoded at least five genes in each of the three other sub-families (CE5, cutinase; CE11, UDP-3-O-acyl N-acetylglycosamine deacetylase; CE12, GDSL-like lipase/acylhydrolase; and CE13, pectinacetylesterase).Fewer CE14 (GlcNAc-PI de-N-acetylase) genes were found inPhytophthora; for example, no CE14 gene was identified inP.cinnamomi,P.capsiciandP.lateralis.At the orthologous group level, genes belonging to four groups (PIGROUP1351 and PIGROUP1376 in CE8; and PIGROUP1152 and PIGROUP3921 in CE13) were found in all eightPhytophthorastrains (Appendix E). Additionally,27 groups of four sub-families (CE5, CE8, CE12 and CE13) contained no genes inPhytophthora, indicating specific roles for genes in these families in species of theHyaloperonosporaandPythiumgenera.

Table 2 Carbohydrate esterase (CE) genes predicted by two methods, CAZymes Analysis Toolkit (CAT, threshold of 1e-50) and dbCAN (default parameters)

In the analysis, abundant genes putatively involved in hemicellulose and pectin degradation were identified inPhytophthora. Three sub-families, CE8, CE12, and CE13,are involved in pectin degradation (Martens-Uzunova and Schaap 2009). The roles of pectin-methylesterases(CE8) and pectin-acetylesterases (CE12 and CE13) are to remove methyl groups from α-1,4-galacturonic acid residues and acetyl groups from galacturonic acid residues in pectin, respectively (Coutinho 1999; van den Brink and de Vries 2011; Blackmanet al. 2014; Sénéchalet al.2014). The existence of the abundant genes encoding these enzymes in some groups suggest their functional significance during plant infection.

Table 3 A total of 445 carbohydrate esterase (CE) genes identified by PLdcg (pipeline for discovering CE genes)

Fig. 2 The distribution of carbohydrate esterases (CEs) in 16 oomycete genomes. A, the phylogeny of the 16 genomes constructed based on the amino acid sequences of 111 single-copy genes using Tree-puzzle with the Dayhoff Model. Circle sizes visually represent the followed counts of CE genes of the genomes. B, different color depth blocks correspond to different counts of sub family of CEs genes in different oomycete species.

3.4. Phylogenetic analysis of CE13 genes

The pectinacetylesterase-associated proteins include putative reductases, transferases, hydrolases,ribonucleases, oxidases, kinases, and other proteins,as well as 14 hypothetical proteins (Appendix K),demonstrating a wide functional diversity. While the losses of associated proteins in somePhytophthoraspecies include an aspartate aminotransferase (PITG_02256), a NAD kinase (PITG_02750), a sulfite reductase [NADPH]flavoprotein (PITG_19263), a transketolase (PITG_01752),a phosphoglycerate mutase (PITG_07400), a cyclopropanefatty-acyl-phospholipid synthase (PITG_02277), and a Na/H antiporter (PITG_03966) (Appendix K). Some of these proteins may also be involved in pathogenesis, including the Na/H antiporter, because Na/H antiporters, membrane proteins have a major role in pH and cellular ion homeostasis(Padanet al. 2001), and their members are essential for virulence of the zoonotic pathogenYersinia pestis(Minatoet al. 2013). In addition, for three hypothetical proteins(PITG_06003T0, PITG_07249T0 and PITG_21054T0), GO analysis suggested their functions in binding, catalytic, or transporter activities, respectively (Fig. 4-B), improving the annotation of these associations.

云計(jì)算按照服務(wù)類型大致可以分為IaaS、PaaS和SaaS三類[3]。IaaS給用戶提供所有計(jì)算機(jī)基礎(chǔ)設(shè)施的利用，如Salesforce客戶關(guān)系管理（Salesforce CRM）。PaaS將采用提供的開發(fā)語言和工具（如Java、Python、.Net等）開發(fā)的應(yīng)用程序部署到應(yīng)用上的云基礎(chǔ)設(shè)施上去，如谷歌搜索引擎。SaaS提供給用戶的服務(wù)是運(yùn)營商運(yùn)行在云計(jì)算基礎(chǔ)設(shè)施上的應(yīng)用程序，用戶可以在各種設(shè)備上通過客戶端界面訪問，如亞馬遜C2和亞馬遜S3。

Fig. 3 Phylogenetic analysis of CE13 subfamily genes and representations of gene loss and duplication events. A neighborjoining tree of CE13 genes was generated using the p-distance model implemented in TreeBeST (boostrap 1 000). The majority of CE13 sequences contained a single pectinacetylesterase domain; however, two genes, PAG1_06709 and PVE_02897, had two pectinacetylesterase domains (indicated by rectangles). The 58 (84.06%) secreted proteins were marked in bold. And six units of gene clusters were marked by blocks. Each gene unit represented a species tree like topology.

3.5. The diversity of pectinacetylesterase associations in Phytophthora

A functional association network forP.infestansgenerated by a Bayesian approach that integrated interacting proteins,co-expressed, and co-occurring proteins, was analyzed to investigate previously reported complex, systemlevel biology (Seidlet al. 2013), providing a potential data resource for comparison of protein associations inPhytophthora, which will help in understanding the pathology and evolution of these pathogens. Proteins associated with CEs ofP.infestanswere selected for comparative analysis in this study. These include 230 proteins, forming eight single-core subnetworks (Appendices I–J). In particular, the PITG_14270 (pectinacetylesterase) subnetwork contained 54 proteins (23.5%). Similar to previous studies (Lehner and Fraser 2004; Leiet al. 2014), we identified potential protein associations in otherPhytophthoraspecies using the InParanoid algorithm (?stlundet al. 2010) with an interaction score cutoff value of 0.5 (Appendix K). Confident associations with pectinacetylesterases were identified for sevenPhytophthoraspecies (Fig. 4-A). For the 54 associations between PITG_14270 (a secreted protein)and other proteins (Fig. 4-B; Appendix K), some of the associated partners were lost in other species, including PITG_14270-PITG_19263, PITG_14270-PITG_02598,PITG_14270-PITG_04104, and PITG_14270-PITG_07155,which are absent fromP.capsici, a species closely related toP.infestans. A minimum of two (3.70%) associations were absent in bothP.sojaeandP.ramorum, while a maximum of 27 (50.00%) associations were absent inPhytophthora cinnamomi; In addition, 37 (68.52%)associations with pectinacetylesterases were absent in at least one of the seven genomes (Fig. 4-B; Appendix K). These results suggest dynamic changes in pectin catalysis inPhytophthoraduring evolution. Commonly absent associations in fourPhytophthoraspecies were discovered for the hypothetical proteins PITG_14270-PITG_02598, PITG_14270-PITG_04104, and PITG_14270-PITG_07155 (Fig. 4-B). Four homologous associations(PITG_14270-PITG_17921, a putative P-type ATPase superfamily protein; PITG_14270-PITG_01752, a putative transketolase; PITG_14270-PITG_07400, a putative 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase; PITG_14270-PITG_22562, a putative croquemortlike mating protein) were expanded in two (P.nicotianaeand theP.sojae) genomes (Fig. 4-B; Appendix K). No other expansions were identified inPhytophthoragenomes.

將壓縮機(jī)管網(wǎng)的正常出口壓力設(shè)置為P1，將現(xiàn)場實(shí)際測定的壓力值設(shè)置為P2，值由PLC中PID功能模塊進(jìn)行統(tǒng)計(jì)、計(jì)算，根據(jù)控制變頻器的具體情況對電動(dòng)機(jī)的運(yùn)轉(zhuǎn)速度進(jìn)行合理化的調(diào)整，從而實(shí)現(xiàn)規(guī)定的具體的壓力。ΔP值與現(xiàn)場壓力、變頻器輸出頻率、電動(dòng)機(jī)的運(yùn)轉(zhuǎn)速度成正比，當(dāng)ΔP＞0 時(shí)，其相關(guān)因素會(huì)隨之提高，風(fēng)壓也會(huì)逐漸升高；當(dāng)ΔP＜0 時(shí)，其相關(guān)因素會(huì)隨之降低；不斷的調(diào)整，使ΔP趨于0，保證壓力處于穩(wěn)定的狀態(tài)，具體系統(tǒng)結(jié)構(gòu)見圖1。

Eight CE sub-families were identified in this study; however,the CE13 subfamily is particularly useful for investigation of genetic evolution, for three reasons: (i) the abundance of CE13 genes; they comprise 15.51% (69/445) of all identified CEs; (ii) more CE13 genes were identified inPhytophthorathan in other genera (Wilcoxon rank sum tests,P＜0.01); (iii)the important roles of pectinacetylesterases during plant infection. Therefore, we performed phylogenetic analysis and identified gene structure variations in CE13 genes, to facilitate the understanding of CE evolution.

4. Discussion

“我覺得是壞事，”他點(diǎn)點(diǎn)頭，“嗯，絕對是壞事，現(xiàn)在已經(jīng)夠難受了?！彼贮c(diǎn)點(diǎn)頭，好像在確認(rèn)剛才說的話，然后轉(zhuǎn)身離去。

Fig. 4 Analysis of pectinacetylesterase associations in Phytophthora. A, homologous genes of PITG_14270 (a secreted pectinacetylesterase) were identified in seven other species. Pin, Phytophthora infestans T30-4; Pca, Phytophthora capsici LT1534;Pso, Phytophthora sojae P6497; Pra, Phytophthora ramorum Pr102; Pla, Phytophthora lateralis MPF4; Pni0, Phytophthora nicotianae race 0; Pni1, Phytophthora nicotianae race 1; Pci, Phytophthora cinnamomi. B, associations (such as PITG_14270-PITG_09284)between Phytophthora infestans pectinacetylesterases and their homologs in other species (such as HJ0_GLEAN_10012171-HJ0_GLEAN_10014439, a homologous pair genes of PITG_14270-PITG_09284 in Phytophthora nicotianae race 0 as detailed gene information shown in Appendix K; and no homologous genes of PITG_09284 found in Phytophthora cinnamomi). Among the 54 associations, 17 were found in all eight genomes. Missing homologous genes are displayed as unfilled circles. The occurrence of multiple homologous genes is represented by multiple circles (such as HJ0_GLEAN_10012385 and HJ0_GLEAN_10004400, the two homologous genes of PITG_17921 found in P. nicotianae race 0). Gene ontology (GO) annotations for associated proteins are shown (such as PITG_09284 annotated by three kinds of annotations, including ‘binding’, ‘catalytic activity’, and ‘structural molecular activity’).

The 16 selected strains included in these analyses represent three lifestyles: hemibiotroph, obligate biotroph,and necrotroph (Baxteret al. 2010; Lévesqueet al. 2010),which raises the significant question of how these different lifestyles arose during evolution? Previous research has demonstrated that horizontal gene transfer events allowed oomycete strains to adapt to new lifestyles (Savoryet al.2015). And the horizontal gene transfer hints for CE5 inPhytophthorahad been discovered (Belbahriet al. 2008).A comparison of gene family sizes between organisms can explain the variation in loss and gain events in the particular gene families, and these events are thought to be directly correlated with the lifestyle and specific adaptations of the organisms (Martenset al. 2008). For example, the massive loss of genes involved in basic processes such as amino acid, carbohydrate, and lipid metabolism reflect the transition from a free-living to an obligate intracellular lifestyle in theApicomplexa(Martenset al. 2008). In addition, amongPhytophthora, expansions of species-specific genes have been proven to have a role in the degradation of plant cell wall polysaccharides to facilitate pathogen invasion (Jianget al. 2006; Ospina-Giraldoet al. 2010a). Our results suggest a relationship between hemibiotrophic strains and abundant CEs in three subfamilies (CE8, CE12, and CE13),by comparison with strains with the two other lifestyles(biotroph and necrotroph) (Fig. 2), suggesting that pectinand hemicellulose-degrading enzymes closely correspond with the hemibiotrophic lifestyle. Therefore, the diversity of lifestyle-related functional proteins may be a consequence of species adaptation to their environments and hosts.

本公路工程攤鋪機(jī)的施工建設(shè)采用了弗格勒2100攤鋪機(jī)，因其無法作用于透水瀝青路面的邊角部位，應(yīng)以人工作業(yè)方式進(jìn)行攤鋪、壓實(shí)以及找平的處理。對于攤鋪厚度的控制，應(yīng)將系數(shù)確定為1.05，以為單位面積內(nèi)攤鋪混合料重量的控制，進(jìn)而提高施工作業(yè)的精度與質(zhì)量。此外，還應(yīng)與拌和裝置相配合以保證混合料提供的連續(xù)性，并杜絕橋面鋪裝層上的緊急轉(zhuǎn)彎與調(diào)頭作業(yè)，來強(qiáng)化工程項(xiàng)目的施工建設(shè)質(zhì)量。

4.4 營造良好的醫(yī)療護(hù)理安全文化氛圍 為了創(chuàng)造寬松、溫馨的醫(yī)療環(huán)境，有效緩解臨床護(hù)士心理壓力，可利用院內(nèi)網(wǎng)絡(luò)資源建立護(hù)理不良事件園地、護(hù)理部主任信箱等信息溝通平臺(tái)，讓臨床護(hù)士的真實(shí)想法得到暢所欲言，為護(hù)士提供一個(gè)共享、平等、快捷且自由的網(wǎng)絡(luò)信息資源，鼓勵(lì)護(hù)士自愿上報(bào)，加強(qiáng)整個(gè)系統(tǒng)的保密性，營造一種“安全文化”的氛圍，把不良事件上報(bào)的管理制度提升到文化管理的層次，放棄目前拒絕承認(rèn)錯(cuò)誤，懲罰失敗的文化，使醫(yī)院每位護(hù)理人員在正確的安全觀念支配下規(guī)范自己的行為［10］。

5. Conclusion

We proposed a pipeline to discover CEs. We identified CEs in oomycetes and discovered significantly more CEs inPhytophthorathan inHyaloperonosporaandPythium,supporting by the phylogenetic analysis of CE13 genes.Additionally, we discovered the evolutionary diversity of CE genes, which may facilitate exploration of CE evolution in pathogens.

Acknowledgements

This work was supported by the Special Fund for Agro-scientific Research in the Public Interest, China(201303018), the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences(CAAS-ASTIP-IVFCAAS) and the emarked fund for the China Agriculture Research System (CARS-25-B-01).

除了音樂和對白，其余聲音效果的總稱就是動(dòng)效。動(dòng)效包括機(jī)械音響和動(dòng)作音響。動(dòng)效制作技術(shù)能夠增加作品的真實(shí)性，對畫面效果具有很大的影響。在制作動(dòng)畫的過程中，動(dòng)效制作技術(shù)能夠優(yōu)化細(xì)節(jié)處理，使作品更具趣味性與生動(dòng)性。動(dòng)效制作技術(shù)包括兩個(gè)方面。首先是環(huán)節(jié)設(shè)計(jì)，錄音師要基于整體考慮，將各個(gè)動(dòng)效環(huán)節(jié)設(shè)計(jì)好，進(jìn)行精細(xì)的布局。錄音師要對畫面內(nèi)容有充分的了解，在其中融入自己的巧妙構(gòu)思，提高作品的聽覺藝術(shù)性。其次是過程控制，這一環(huán)節(jié)是動(dòng)效制作真實(shí)性的保障，制作者要立足于生活，對生活細(xì)節(jié)進(jìn)行觀察和推敲，讓作品更有畫面感和層次感，使觀眾獲得更好的聽覺體驗(yàn)。

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