Nematophagous (NP) fungi are ecologically important components of the soil microbiome in natural ecosystems. Esteya vermicola (Ev) has been reported as a NP fungus with a poorly un- derstood evolutionary history and mechanism of adaptation to parasitism. Furthermore, NP fun- gal genomic basis of lifestyle was still unclear. We sequenced and annotated the Ev genome (34.2 Mbp) and integrated genetic makeup and evolution of pathogenic genes to investigate NP fungi. The results revealed that NP fungi had some abundant pathogenic genes corresponding to their niche. A number of gene families involved in pathogenicity were expanded, and some pathogenic orthologous genes underwent positive selection. NP fungi with diverse morphologi- cal features exhibit similarities of evolutionary convergence in attacking nematodes, but their genetic makeup and microscopic mechanism are different. Endoparasitic NP fungi showed simi- larity in large number of transporters and secondary metabolite coding genes. Noteworthy, ex- panded families of transporters and endo-beta-glucanase implied great genetic potential of Ev in quickly perturbing nematode metabolism and parasitic behavior. These results facilitate our understanding of NP fungal genomic features for adaptation to nematodes and lay a solid theo- retical foundation for further research and application. Key words: nematophagous fungi, Esteya vermicola, comparative genome, genome feature, adaptation and evolution 1. Introduction xylophilus), which exhibits high infectivity. Certain volatile organic The pine wood nematode is responsible for an epidemic of pine wilt compounds (VOCs), a-pinene, b-pinene, and camphor for PWN at- disease, which causes severe ecological and economic losses in Asia traction were produced by Ev. Then lunate adhesive spores produced 1,2 and Europe and more recently spreading to Mexico. Esteya vermi- by Ev, whose concave surface has large surface area and thick adhesive cola (Ev) is the ﬁrst recorded endoparasitic nematophagous (NP) layer, adhered to the cuticle of attracted nematode, and penetrated the fungus that attacks the pine wilt nematode (PWN; Bursaphelenchus cuticle of PWN. Afterwards, Ev colonized in the body cavity of the V The Author(s) 2018. Published by Oxford University Press on behalf of Kazusa DNA Research Institute. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact firstname.lastname@example.org 245 Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/3/245/4791394 by Ed 'DeepDyve' Gillespie user on 26 June 2018 246 Genome features of nematophagous fungi Figure 1. Life cycle of the Ev. (a) hyphae and lunate spore (arrow); (b) nematodes were attracted and adhered by lunate spores (arrow); (c) absorption of nutri- tion from nematode corpse, and producing new lunate spores (arrow); (d) emergence of new hyphae from the nematode; (e), mass reproduction of hyphae and spores on the nematode; f, attracting and infecting nematodes again. The whole infection process about 48 h. Scale bar, 50 mm; inset in a, 5 mm. The infected nematode shown is B. xylophilus. nematode and produced a great deal of mycelia and lunate spores on underpinnings underlying the establishment of predation relationship cadaver of nematode to attract new nematodes for next infection cycle in NP fungi by performing comparative genomic and evolutionary (Fig. 1). Once lunate spore adheres to nematode cuticle, spores fall-off analysis. Here, we reported the complete genome sequence of Ev from the conidiophores and move with nematode. Ninety percent of based on the PacBio sequencing technology, comprehensively ana- 3,6 inoculated PWN are infected within 24 h. The high infectivity sug- lyzed the differences among NP, PP, and IP fungi, and uncovered the gests that Ev has great potential as biocontrol agent against PWN, but genetic and evolutionary bases of NP fungi (Ao, Dh, Dc, Hm, Pc and the evolution and mechanisms underlying infection are not clear. Ev) by the numbers of pathogenic genes, secreted protein, secondary NP fungi are important microorganisms that can suppress the metabolism encoding genes, expanded gene families, and genes un- populations of plant-parasitic nematodes and maintain ecological dergoing positive selection. balance and can be used as potential biological control factors. The genomes of NP fungi which have been sequenced include Arthrobotrys oligospora (Ao) with adhesive networks, Dactylellina 2. Materials and methods haptotyla (Dh; synonyms Monacrosporium haptotylum) of knob- 2.1. Fungal strain and DNA preparation 9 10,11 forming species, Drechmeria coniospora (Dc), Pochonia Ev (CBS115803) was purchased from CBS and maintained on corn- 12,13 chlamydosporia (Pc) and Hirsutella minnesotensis (Hm)of meal agar. This fungus was originally isolated from Scolytus intrica- endoparasitic fungus, and Drechslerella stenobrocha (Ds) with tus and its galleries in oak trees in Czech Republic. Mycelium was constricting ring. Despite the important roles of NP fungi in ecol- grown in liquid medium (potato dextrose broth) and incubated at ogy, comprehensive and detailed analysis of similarity and difference 25 C using a shaker at 150 rpm for 7 days. The mycelium (2g) was based on the multiple genomic level was still lacking. Due to very dif- harvested by aseptic ﬁltering and then grounded in liquid nitrogen. ferent hosts categories and lifestyles of insect pathogenic (IP), plant Genomic DNA was extracted using Qiagen Genomic-tip Kit 500 G pathogenic (PP) and NP fungi, it has been expected that their capacity (Cat No./ID: 10262, Germany) according to the manufacturer’s in- to utilize the host’s nutrition and to degrade carbohydrates and proteins structions. DNA quality was assessed by spectrophotometry and gel would also differ, and that might be reﬂected in their genetic makeup electrophoresis before library construction. and gene copy numbers at ﬁrst. The availability of multiple whole ge- nome data of NP, IP, and PP fungi has provided good opportunity to 2.2. SMRT PacBio sequencing and HGAP genome employ genome-wide surveys to investigate how natural selection shaped the evolution of NP fungi towards nematode host. assembly The study aimed to decipher the putative genetic components in- Twenty-ﬁve micrograms of high-molecular-weight Ev gDNA was volved in pathogenesis and evolution and to identify the molecular used for the PacBio libraries constructing. A 20-kb insert SMRTbell Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/3/245/4791394 by Ed 'DeepDyve' Gillespie user on 26 June 2018 R. Wang et al. 247 library was generated using a 15 kb lower-end size selection protocol TCDB, DFVF, and VFDB) were evaluated by correspondence analy- on the BluePippin (Sage Science). The genome was sequenced using 4 sis. All statistical analyses were performed with R project V.3.4.2. SMRT Cells and P6-C4 chemistry on the PacBio RS II platform (Paciﬁc Biosciences). The Ev genome was assembled using the HGAP 2.5. Phylogeny construction and estimation of (version 2.3.0). First, the genome was preassembled with improved divergence time consensus accuracy. Then it was assembled through overlap consen- Twenty-three fungi species [Ev, Dc, Ao, Dh, Hm, Pc, Gc, Op, Ss, sus accuracy using Celera assembler and polished with Quiver to im- Sporothrix brasiliensis (Sb), Fusarium graminearum (Fg), prove the site-speciﬁc consensus accuracy of the assembly. This Magnaporthe oryzae (Mo), Colletotrichum higginsianum (Ch), Whole Genome project has been deposited at SRA database in NCBI Verticillium dahliae (Vd), Zymoseptoria tritici (Zt), Cordyceps mili- under the accession SRP097009 (BioProject: SUB2318437; taris (Cm), Beauveria bassiana (Bb), Metarhizium anisopliae (Ma), BioSample: SUB2318464). Metarhizium robertsii (Mr), Moelleriella libera (Ml), Sporothrix insectorum (Si), Neurospora crassa (Nc), Saccharomyces cerevisiae 2.3. Gene prediction and functional annotation (Sc)] were used for gene family clustering and phylogeny tree con- Gene annotation was conducted by combining de novo and struction. Blastp was used to generate the pairwise protein sequence 18 19 5 34 homology-based methods. Augustus and GeneMark-HMM were with similarity (E-value 1e ). OrthoMCL was used to cluster employed to de novo predict gene structures in the Ev genome, re- similar genes by setting main inﬂation value 1.5 and other default pa- spectively. For homology-based prediction, protein sequences from rameters; 1203 single-copy gene families were extracted for the phy- three species [Grosmannia clavigera (Gc), Ophiostoma piceae (Op), logenomic analysis. The protein sequences were aligned by Mafft Sporothrix schenckii (Ssch)] were initially mapped onto the Ev ge- and back translated into CDS alignments. Then, we eliminated nome using tBlastn (E-value 1e ). The homologous genome se- poorly aligned positions and divergent regions of alignment of CDS quences were aligned against the matching protein using GeneWise sequences using Gblocks. Four-fold degenerate sites of all these for accurate spliced alignments. All the predictions were combined single-copy genes in each species were extracted and concatenated by EVidenceModeler (EVM) to produce a consensus gene sets. them to be one supergene for phylogeny construction. Phylogenetic Gene functions were assigned according to the best matches derived analyses using maximum likelihood (ML) method were conducted in from the alignments to proteins annotated in KOG, NR, SwissProt, RaxML version 8.2.4 and set Sc as out-group. The divergence time 22 5 and TrEMBL databases using Blastp (E-value 1e ). Then the was estimated based on established phylogeny tree. Markov chain pathway in which the gene might be involved was annotated by Monte Carlo algorithm for Bayes estimation was adopted to estimate KAAS according the KEGG database. Motifs and domains were the neutral evolutionary rate and species divergence time using the annotated using InterProScan by searching against GO databases. program MCMCTree of the PAML package. The calibration times Finally, the result annotated from the KOG, GO, KEGG, NR, for divergence between Sc and Dh (460-726My), Dh and Zt (358- Swissprot, and TrEMBL databases were combined to obtain the ﬁnal 468Mya), Pc and Ch (194-888Mya) were obtained from the annotation of Ev genome. TimeTree database. 2.4. Protein family classifications and prediction of 2.6. Expansion and contraction of gene families pathogenicity-related genes Gene families that have undergone expansions or contractions were The carbohydrate active enzymes (CAZy) genes were identiﬁed using identiﬁed using CAFE version 3.0 program with default parame- Blastp according to the CAZy database. Whole genome protein ters. The algorithm CAFE takes a matrix of gene family sizes in ex- families were classiﬁed by Pfam analysis. The families of proteases tant species as input and uses a probabilistic graphical model to were identiﬁed by Blastp searching against the MEROPS peptidase ascertain the rate and direction of changes in gene family size across database release 9.4. Transporters were retrieved from the the given phylogenetic tree. The tree and expansion/contraction data 28 41 Transporter Classiﬁcation Database (TCDB). Putative virulence were displayed using the iTOL web tool. factors were identiﬁed by searching against the pathogen–host inter- action database (PHI), database of virulence factors in fungal path- 2.7. Identification of orthologous genes and positively ogens (DFVF), and virulence factors of bacterial pathogens selected genes (PSGs) (VFDB). Peroxibase-encoding genes were predicted searching against Orthologous genes in 23 species were identiﬁed. Genes with CDS Peroxidases database. Antibiotic resistance genes were identiﬁed ac- length less than 450 bp were ﬁltered. All-versus-all Blastp compari- cording to Antibiotic Resistance Genes Database (ARDB). An E- sons were used to identify clusters of homologous genes. For avoid- value cut-off of 1.0E-5 was adopted to ﬁlter the BLAST results of the ing noise information in the Blastp output, orthologous clusters that above databases. Twenty-one strains of fungi were used for the contained more than 10 species were retained. The values of Ks and above BLAST analysis (Supplementary Table S1). SignalP 4.1 and Ka substitution rates and the Ka/Ks ratio were estimated using the TargetP were used to predict potential secreted proteins with default Codeml program in the PAML package. Codeml was used to cal- parameters. Secondary metabolite genes of six NP fungi, six IP, and culate the selection pressure in the phylogenetic tree (P 0.05). six PP fungi were predicted by SMURF (Supplementary Table S1). The antiSMASH pipeline with HMM signatures was used to identify and annotate putative polyketide synthase (PKS), nonribosomal pep- 3. Results tide synthetase (NRPS), and terpene synthase (TPS) genes and other 3.1. General features of Ev genome gene clusters about secondary metabolites and to predict the PKS and NRPS domain architecture. The whole genome dataset was sub- Ev genome was sequenced at approximately 100 coverage, gener- jected to analysis with default settings. The correlations between ating a total of 4.44 Gbp sequences. De novo assembly yielded Ev these 18 fungal species (IP, PP, and NP) and gene variables (PHI, genome of 34.2 Mb in length with average 68.59 coverage depth. Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/3/245/4791394 by Ed 'DeepDyve' Gillespie user on 26 June 2018 248 Genome features of nematophagous fungi The N50 sizes of 50 contigs and 42 scaffolds were 4.26 Mbp and G-protein receptor (Supplementary Fig. S12), pectate lyase 4.41 Mbp, respectively. Combined de novo and homology prediction (Supplementary Fig. S13) and cutinase (Supplementary Fig. S14), produced 8,427 protein-coding genes. The detailed annotation re- P450 (Supplementary Fig. S15). The important claim here was that the sults are given in the supporting information. number of hemicellulose, G-protein receptor, and pectate lyase in PP fungi was signiﬁcantly higher than that in IP fungi (Supplementary Figs S12b and S13b). Moreover, the content of cutinase in PP group 3.2. Comparative analysis of gene involved in was signiﬁcantly greater than that in IP and CP group (Supplementary pathogenicity and virulence Fig. S14b). It suggested that PP fungi evolved to adapt to higher carbo- To identify functional gene categories characteristic of adaptation in hydrate content in plants. killing nematode, we compared 21 genomes, including 6 IP fungi, 6 We identiﬁed 231 transporter superfamily in all 21 fungi species. PP fungi, and 6 NP fungi and 3 genetically close species with Ev, Gc, Ev, Hm and Pc genome encodes a large number of transporters and Si (Supplementary Table S1). The genome integrity and com- (1,482, 1,546, and 1,986, respectively; Supplementary Table S10). pleteness of gene prediction assessed by BUSCO are shown in Remarkably, Ev and Pc had more carbon (72, 90), iron (23, 19) and Supplementary Table S2. The genome integrities of the selected spe- vitamin transporters (81, 55) than do other NP fungi (Fig. 2 and cies were more than 95%, and the completeness of protein prediction Supplementary Table S10). The numbers of genes encoding allantoate was greater than 92%. The corresponding datasets based on differ- transporters, and vitamin or iron in Ev were the greatest and second ent databases were shown in supporting information (Supplementary highest among all 21 fungi, respectively (Fig. 2). Ammonium and urea Table S3–S11). Extracellular adhesive proteins comprise GLEYA import capacity of Ev was higher than other NP, IP, and PP fungi. proteins, CFEM proteins, WSC-containing proteins and lectin, each Therefore, Ev shows an increased genetic potential in terms of vitamin, of which was averagely the highest in NP genomes except CFEM iron, and nitrogen uptake. This could be an important mechanism for (Supplementary Fig. S1), as well as total adhesion (Supplementary perturbing nematode metabolism and ultimately causing nematode Fig. S2). Dh encoded the highest number of adhesive proteins death. It indicated that the transport capacity of ammonium, nucleo- GLEYA and WSC, whereas Ao encoded most abundant lectin base, and nucleoside in CP group was very strong (Supplementary Fig. among 21 analyzed fungi genomes (Supplementary Fig. S1a). S16). The number of oligopeptide transporter in IP group was aver- However, our comparative analysis found that endoparasitic fungi agely higher than other groups and signiﬁcantly higher than that of in (Ev, Dc, Pc and Hm) contain fewer extracellular adhesive proteins NP group (Supplementary Fig. S16). This implied the great potential of (Lectin and GLEYA) than adhesive networks producer Ao and adhe- absorbing oligopeptide of nitrogen sources in IP fungi. sive knobs producer Dh (Supplementary Figs S1 and S2). It was Strikingly, all 21 fungi contained a wide array of antibiotic resis- found that subtilases/peptidase S8 and subtilisin-like participating in tance genes. The total number of antibiotic resistance genes in IP 42–44 lethal activity and the infection process of nematodes were aver- fungi (207) was the richest, followed by PP fungi (201), and NP fungi agely abundant in NP (Supplementary Fig. S3 and Supplementary (174) (Supplementary Table S8 and Supplementary Fig. S17). The Table S6). More subtilisin S8 and subtilisin-like were found in Ao substances encoded by these genes were likely to play roles in compe- and Dh than that in other NP fungi (Supplementary Table S6). tition with bacteria in insect, nematode, and plant, then pathogenic NP fungi encoded averagely richer ankyrin repeat protein, while PP fungi can colonize in their host. Among antibiotic synthesizing genes, fungi encoded more leucine-rich repeat, tyrosinase, xylanase macrolide transporter-encoding genes and multidrug-encoding genes (Supplementary Fig. S4). IP fungi encoded the least cellulose, com- were most abundant in each group (Supplementary Table S8 and pared with PP, NP and CP fungi (Supplementary Fig. S6). Supplementary Fig. S17). Interestingly, all analyzed fungi except Z.tritici possessed appresso- rial penetration protein (PHI: 256), homologs of proteins in plant- 3.3. Secreted proteins and small secreted proteins pathogenic fungi Mo, especially in Ao (25) and Dh (28) most abundant (Supplementary Table S4). Secretion signals were predicted in 21 fungi. The results showed that Chitinase is important for IP and egg-parasitic fungi to infect insect PP had the highest number of secreted proteins (SPs) and small se- 45,46 and nematode eggshell. IP fungi were most abundant averagely creted proteins (SSPs;< 300 amino acids), followed by NP and IP in enzymes catalyzing the decomposition of chitin (Supplementary fungi (Supplementary Table S12). Among the 21 analyzed fungi, Mo Fig. S7). Pc, an egg-parasitic NP fungus, encoded the highest number had amazing ability to produce SSP (about one-tenth of the total pro- of chitinase (159, CAZy) among NP fungi (Supplementary Fig. S7). tein). Among NP fungi, Pc had the most SPs and SSPs, followed by Chitosanases (including GH5, GH7, GH8, GH46, GH75 and GH80) Dh, Ao and Hm (Supplementary Table S12). Out of the 1,318 proteins are another enzymes involved in chitin degradation and nematode predicted to be secreted by Ev, 46.7% can be ascribed known function. parasitism. The analysis suggested that Ev had the most chitosa- Of these 397 SSPs of Ev, 302 was annotated into NR database, but nases, especially GH7; Ao encoded the richest GH75 (Supplementary only 131 (33.0%) were annotated with known function; 10 of 397 Table S3 and Supplementary Fig. S7). IP, NP, and PP fungi also SSPs was related with transporters; 48 of 397 SSPs (12.1%) was re- contained averagely more peptidase than CP fungi (Supplementary lated with pathogen-host interaction based on PHI database; and 180 Fig. S8). IP fungi possessed most pkinase and enterotoxin-a, further- of 397 SSPs (45.3%) was found listed in Pfam database. more, and both pkinase and enterotoxin-a in IP fungi was signiﬁcantly The possible pathogenic-related SPs are shown in Fig. 3. NP fungi higher than that in PP fungi (Supplementary Figs S9 and S11). Based were rich in SPs of subtilisin or subtilisin-like protease, lectin, WSC, on Pfam database, IP had signiﬁcantly more toxin genes than PP and iron transporter. Only Ev was predicted to produce secretory and CP fungi (Supplementary Fig. S10). The number of aspartic cellobiose dehydrogenase in NP fungi. Ev was remarkable in SPs of peptidase in NP and IP fungi was averagely higher than putative aspartic protease, glucanase and endoglucanase, laccase and PP fungi (Supplementary Fig. S5). PP fungi genome encoded the richest cellobiose dehydrogenase. It was worth noting that the number of se- CAZy enzymes (Supplementary Table S3), peroxidase (Supplementary creted oligopeptide transporter and aspartic proteinase was remark- Table S9), in particular, hemicellulose (Supplementary Fig. S12), ably high in endoparasitic fungi Ev, Dc, Pc, and Hm. The important Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/3/245/4791394 by Ed 'DeepDyve' Gillespie user on 26 June 2018 R. Wang et al. 249 Figure 2. Heatmap of vitamin, iron, saccharides and nitrogen transporter-encoded genes in 21 fungi. Figure 3. Heatmap of possible pathogenic related SPs in 21 fungi. SPs involving pathogenicity in Ev were comparable to those in Mo genes in Pc (195, 156) and Hm (299, 238) were more abundant than and Ch, such as P450, laccase, and cellobiose dehydrogenase. that of other analyzed NP fungi. Especially endoparasitic NP fungi had tremendous potential for biosynthesis of secondary metabolites that act as toxins or signals in the interactions between fungus and 3.4. Secondary metabolite analysis host. The rich unknown secondary metabolism in NP fungi is worthy PP fungi can produce diverse secondary metabolites (SMs) that aid in of further research and exploration. Furethermore, Mr and Ma of IP 47–49 pathogenicity. However, the extent and distribution of the ca- fungi, and Ch and Mo of PP fungi also had great capacity for biosyn- pacity for secondary metabolite synthesis have not been investigated thesis of secondary metabolites, which possibly greatly enhanced in NP group. Furthermove, there was no comparative analysis about their disease-causing abilities. the capacity for secondary metabolite among groups (IP, NP, and PP) before. SMURF predicted that endoparasitic fungi (Hm, Pc, Dc, 3.5. Insights into pathogenic differentiation leading to and Ev) had more genes encoding secondary metabolites than species different hosts categories of adhesive networks and knob-forming species (Ao and Dh) (Supplementary Table S13). Hm had the highest number of gene To assess pathogenic differentiation leading to different host classiﬁ- clusters and core genes involved in biosynthesis of secondary metab- cation, we performed correspondence analysis based on multiple olites, followed by Pc among NP group. PKS and NRPS synthesis large datasets (Supplementary Tables S3–S11). It was hypothesized Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/3/245/4791394 by Ed 'DeepDyve' Gillespie user on 26 June 2018 250 Genome features of nematophagous fungi that pathogenic fungi belonging to the same host categories with sim- closely related, and the divergence of the two species took place ilar nutritional mode, would be clustered together. Figure 4 shows around 212 Mya. Dc, Pc and Hm had a close genetic relationship separation result of three clusters (PP, IP, and NP) according to gene with IP fungal Metarhizium, which was consistent with previous 10–14 variables of PHI genes, membrane transport proteins (TCDB), fungal phylogenetic analysis results. virulence factors (DFVF), virulence factors of bacterial pathogens (VFDB). The species of IP fungi were clustered except Si species, and 3.7. Gene family and gene family expansion so did the six PP species. However, the distribution of NP species Gene duplication and protein family expansion are important geno- was dispersive, and Ev, Dc and Pc were always clustered with PP, IP mic mechanisms that shape the evolution of pathogenic fungi. fungi, respectively. CAZy, peroxidises, and antibiotic resistance CAFE analysis was performed to assess the gene family variations; genes failed to separate IP and PP fungi (figures not shown). Also, 276 gene families were expanded in Ev, 1,170 families had under- the analysis suggestsed that the difference in pathogenicity genes or gone contraction (Fig. 6). We found a marked expansion of transpor- membrane transport proteins was possibly the important factors to ter genes in Ev. Among the 276 expanded gene families in Ev, drive pathogenic differentiation and adaptation to different host cat- 10.7% (37) was related to transporters or permeases, which was the egories. Endoparasitic fungal Ev, Dc, Pc and Hm, which were very highest among all the fungi analyzed (Supplementary Table S14), different from Ao and Dh, were likely to be the intermediate type be- while the proportions were less than 2.4% and 6.8% in other ﬁve tween the IP and PP fungi. NP fungi and other analyzed species, respectively (Supplementary Table S14). It was surprising that the proportion of expanded gene 3.6. Phylogeny and divergence time estimates families of transporters in Ev was much higher than that of the other OrthoMCL was used to identify gene orthologs. Totally 253,363 analyzed species. The transporters of expanded gene families in Ev genes from 23 species were used for gene family clustering analysis. occurred mainly for the carbon source, nitrogen source, iron and vi- Finally, 20,524 gene families containing 206,300 total genes from tamin transport, such as ABC transporter, sugar transporter, iron 23 species were generated, out of which 1,203 possessed a strict permease, urea transporter, allantoate/allantoale permease, vitamin single-copy orthologous relationship. The phylogenomic tree was H transporter and amino acid permease (Supplementary Table S15), constructed using concatenated nucleotide sequences of 1,203 single- which provide the basis for effectively absorbing nutrition of nema- copy orthologous genes to infer NP fungal phylogenetic relationships todes. Gene families involving in aﬂatoxin efﬂux pump had under- in the context of other fungal taxa. It indicated that Ev diverged gone expansion in Ev (Supplementary Table S16). Gene families from plant-associated Gc about 135 million years ago (Mya), and Ev about endo-beta-glucanase, which catalyse hydrolyzing O-glycosyl and Gc had closer genetic relationship (Fig. 5). The phylogenetic compounds, were substantially expanded in Ev compared with other analysis showed that the relationship of Ao and Dh in NP fungi were fungi species. Important pathogenicity-related gene families were Figure 4. Correspondence analysis grouping pathogenic fungi by host class based on the set of predicted (a) PHI, (b) DFVF, (c) TCDB, (d) VFDB. Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/3/245/4791394 by Ed 'DeepDyve' Gillespie user on 26 June 2018 R. Wang et al. 251 Figure 5. Phylogenetic tree and divergence time estimates. The numbers at nodes in the tree are the divergence times from the present (Million years ago, Mya). The nodes with red dot represent the reliable calibration time of the divergence. Bootstrap values in each node are 100 (not shown in the diagram). Six NP fungi H. minnesotensis, D. coniospora, P. chlamydosporia, E. vermicola, A. oligospora, and D. haptotyla Figure 6. Gene family expansions and contractions. Branch length represents differentiation time. Numbers for expanded (green) and contracted (red) gene families are shown below branches or taxon names with percentages indicated by pie charts. Blue represents the proportion of gene families no changed in the current spe- cies or branch. Orange in pie charts represents the proportion of the gene families combined change across lineages. Six NP fungi are marked with red. Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/3/245/4791394 by Ed 'DeepDyve' Gillespie user on 26 June 2018 252 Genome features of nematophagous fungi founded expanded, including peptidase S41, cellobiohydrolase and expanded greatly in Hm. Sabine Fillinger et al. found trehalose rap- laccase. idly degraded upon induction of conidial germination and was re- The six NP fungi in total shared 3,717 gene families, but no ex- quired for the acquisition of tolerance to a variety of stresses in pansion gene families in common were observed (Fig. 7a and b). Aspergillus nidulans. Compared with other analyzed species, there Surprisingly, we found that Ao and Dh shared the largest set of gene was a major expansion in gene families related to allantoate and families (8,078) through pair comparison, followed by Pc and Hm iron transport, laccase and endoglucanase in Ev (Supplementary with 6,807 gene families. Beyond that, the two species (Pc and Hm) Table S16). The ratio of expansion gene families to total gene with similar nematicidal mechanism (endoparasitism) shared the families in Hm and Pc peaked at an incredible 11.0% and 8.8%, most expanded gene families (132). Substantial differences were ob- respectively, while that in other four NP fungi was no more than 4% served in species-speciﬁc gene families among six fungi (Ev 1,027, (Supplementary Table S17), which partly explained the differences in Hm 650, Ao 244, Dh 223, Dc 168, Pc 1,581) in magnitude. The ra- genomic size of these species. tio of species-speciﬁc gene family to the total gene family was the highest in Pc (16.6%), followed by Ev (14.2%) and Hm 8.1% 3.8. Positive selection (Supplementary Table S17). The ratio of species-speciﬁc expanded gene family to the total expanded gene family was 71–86.8% among Positive selection, acting in the evolution of functionally important NP fungi (Supplementary Table S17). NP fungi contained more gene families, is an important driving force for pathogens in adapta- 52–54 gene families not found in other pathogenic fungi (Supplementary tion to a pathogenic lifestyle and host specialization. Identifying Fig. S18) indicating their unknown functional diversiﬁcation. targets of positive selection will help to annotate the genome func- We further compared expanded gene families in 23 species to de- tionally and to elucidate evolutionary processes. Signatures of posi- tect gene family evolution (Supplementary Table S16). Subtilisin-like tive selection specify functionally important regions of the genome. gene family in NP fungi (Dh and Hm) and IP fungi (Mr) were ex- Based on the established phylogeny and 10,610 high-conﬁdence panded, except that in PP species. Genes encoding chitinase, aspartic orthologues identiﬁed among 23 species, positively selected genes proteinase and Wsc domain protein had undergone expansion in NP (PSGs) of six NP fungi and PP fungal Gc was detected. Dh and Ao and PP group, but not in IP group. Furthermore, peptidase S41 fam- had more PSGs (256, 247) than endoparasitic NP (Ev 234, Dc 102, ily protein exhibited expansion in NP fungi (Ev and Dc) and IP fungi Hm 80, Pc 64) (Supplementary Table S18); 97 PSGs in Ev, this was (Ma). Intriguingly, gene families about trehalose metabolism the highest count among 7 analyzed species, belonged to putative Figure 7. Venn diagrams and selected orthologous genes undergoing positive selection in six NP fungi. (a) Venn diagram of gene families; (b) Venn diagram of expanded gene families; (c) Venn diagram of orthologous genes of positive selection; (d) Selected orthologous genes of positive selection. ‘1’means undergo- ing positive selection; ‘0’means no positive selection. Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/3/245/4791394 by Ed 'DeepDyve' Gillespie user on 26 June 2018 R. Wang et al. 253 PHI genes. Moreover, Ev had the largest number of unique positive proteases as SPs underwent expansion and positive selection in NP selection genes (78) (Fig. 7c). The number of PSGs encoding tran- group. Aspartic protease, which was expressed by many pathogenic scription factors were the most highest in Dh (27) all over analyzed ascomycetes during infection, was subjected to positive selection dur- seven species (Supplementary Table S18). Six NP fungi did not share ing the evolution in NP fungi. Nitrogen permease regulator, as well as orthologous gene undergoing positive selection. Corresponding to some proteolytic enzymes, was under forward selection, implying the the need for nitrogen, orthologous gene (ortholog00083) involving importance of nitrogen sources for NP fungi. Cytochrome P450s play in nitrogen permease regulator have been subject to a positive selec- various roles in secondary metabolism and involve in biodegradation tion pressure in Ev (P ¼ 0.02) to adapt to nematode. Subtilisin-like of lignin and various xenobiotic compounds. Our results indicated proteinase (ortholog00552) was also under positive selection in Ao, that PP and IP contained richer P450s than NP group. Nevertheless, Dh and Hm (Fig. 7d). Orthologous genes related to aspartic endo- P450 and P450 reductase underwent positive selection in NP fungi but peptidase, aminopeptidase, and cytochrome P450 reductase revealed not in PP fungal Gc (Fig. 7d). Therefore, P450s should be functionally positive selection in NP fungi (Fig. 7d). important for NP in adaptation to pathogenic lifestyle. 4.3. Abundance of transporter in Ev genome 4. Discussion We identiﬁed abundant transporters, which underwent expansion in 4.1. NP fungi encoded rich secondary metabolites and Ev. Monosaccharide transporter (Hxt1), also as sensor, is required antibiotics for virulence of the maize pathogen Ustilago maydis and is important Secondary metabolites involving in host–pathogen interactions may for fungal development during the pathogenic stage of the fungus. protect the fungus from predation and allow an organism to survive Pyruvate uptake transporter (BcMctA) is also required for pathoge- 56 57 in its ecological niche. Secondary metabolisms of beauvericin, nicity in Botrytis cinerea, and disruption of BcmctA signiﬁcantly re- 58 59 and bassianolide, and destruxin in insect pathogens were insecti- duced the virulence of B. cinerea on cucumber and tomato leaves. cidal. Similarly, secondary metabolites in NP fungi can kill nema- Phytopathogenic bacteria and fungi can use iron uptake systems to todes. Endoparasitic fungi had very rich repertories of secondary multiply in their hosts and to promote infection, and iron transpor- metabolism genes compared with nematode trapping fungi implying ters have been found to play a critical role in virulence of both ani- endoparasitic fungal great potential for killing nematodes. One of mal- and plant-pathogenic fungi, such as siderophores of the most important questions about the ecology of NP fungi is how Aspergillus fumigatus and high afﬁnity iron permease of Rhizopus 78,79 they defends the nematode cadaver against different microbial com- oryzae, which play critical roles in the expression of virulence. petitors. NP fungi, especially Pc and Hm, encoded a large number of Similarly, these transporters could well take part in pathogenicity polyketide and nonribosomal peptide synthases which could be part during infecting nematodes. In addition, based on a wealth of trans- of the biosynthetic pathway of antibiotics. Besides, the phenomenon porters (Supplementary Table S10), it was possible that NP fungi, es- that NP fungi possess a set of antibiotic resistance genes is a general pecially endoparasitic fungal Ev, Pc, and Hm could be more mechanism responsible for resistance to antibiotics from other mi- efﬁciently utilize nematode nutrients compared with other NP fungi. crobe. It was suggested that antibiotic and fungal secondary metabo- lites displayed antagonistic effect against other microbial competitors 4.4. Endo-beta-glucanase in Ev genome was highly during colonizing nematode cadaver. expanded Comparative analysis revealed endo-beta-glucanase encoding genes 4.2. The abundance and evolution of pathogenic genes in Ev genome were highly expanded. Endo-beta-glucanase likely in NP fungi plays a role in degrading glucans polymers from themselves cell wall Adhesive proteins accumulating on the outside surface of adhesive or their host nematodes. Endo-beta-glucanases were identiﬁed as traps or spores of NP fungi play vital roles for those fungi to adhere 80 parasitism genes in plant-parasitic nematode. Extracellular gluca- to nematode cuticles. Adhesive materials may harbour many se- nase secreted by Acremonium persicinum was considered to partici- creted virulence-related proteins. Comparative analysis showed 81 pate in the degradation of an extracellular storage glucan. that NP fungi contained abundant pathogenicity genes encoding sub- Glucanases produced by some Trichoderma species has been recog- tilisins and subtilisin-like protease, of which the nematotoxic activity nized as the key enzymes in the lysis of cell walls during their myco- has been proved. Transcripts encoding subtilisins were highly ex- 82,83 parasitic action against phytopathogenic fungi. Highly 9,62 pressed by Ao, Dh, and Hm during infection. Previous studies expanded endo-beta-glucanase in Ev genome possibly functions as have proved that subtilisins are important virulence factors involved parasitism, but further research is needed to determine the in the penetration and digestion of host cuticles in IP and NP possibility. 63–65 fungi. We found that tyrosinase-encoding gene was the most abundant present in NP fungi. It has been demonstrated that 4.5. No a- and b-pinene monoterpenoid synthase- tyrosinases involving in synthesis of melanin in some fungi were encoding genes in Ev genome highly upregulated in M. haptotylum (Dh) during infection and participated in pathogenic process. But these genes encoding these Interestingly, the host pine of PWN, vector Monochamus alternatus proteins were not evenly distributed across NP species, and the of PWN, and NP fungal Ev, belonging to three different kingdoms, number of these genes in different species varied widely. These wide all attract nematodes through volatile chemicals: a- and b-pi- 4,7,84,85 differences in number of pathogenic genes may be a manifestation of nene. Due to Ev’ s capacity to attract PWN by volatile chemi- diverse pathogenic mechanism of NP fungi. cals a- and b-pinene and the fact that Ev host intracellular Subtilisin-like proteases as virulence factors function during fungal bacteria, it remains vague whether the fungus itself produced the 67,68 64,69–71 pathogenic progress in IP fungi, NP fungi, and other para- compounds. Annotation results of Ev genome revealed that there site fungi. Our results demonstrated that subtilisin-like serine were no a- and b-pinene monoterpenoid synthase-encoding genes. Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/3/245/4791394 by Ed 'DeepDyve' Gillespie user on 26 June 2018 254 Genome features of nematophagous fungi 7. Li, J., Zou, C. and Xu, J. 2015, Molecular mechanisms of Here we hypothesize that the endobacteria produce the monoterpene nematode-nematophagous microbe interactions: basis for biological con- that attracts PWN. The next step for whole genome sequencing of trol of plant-parasitic nematodes, Annu. Rev. Phytopathol., 53, 67–95. this endobacteria will further verify the authenticity of the endobac- 8. Yang, J., Wang, L., Ji, X., et al. 2011, Genomic and proteomic analyses of teria producing the monoterpenoid that attracts nematodes. the fungus Arthrobotrys oligospora provide insights into nematode-trap formation, PLoS Pathogens, 7, e1002179. 9. Meerupati, T., Andersson, K.-M., Friman, E., et al. 2013, Genomic mech- 5. Conclusions anisms accounting for the adaptation to parasitism in nematode-trapping We sequenced the genome of Ev and embarked upon a comparative fungi, PLoS Genetics, 9, 2005–10. 10. Zhang, L., Zhou, Z. and Guo, Q. 2016, Insights into adaptations to a genomics analysis. These results indicated that NP fungi was distin- near-obligate nematode endoparasitic lifestyle from the ﬁnished genome guished from other pathogenic fungi in terms of pathogenic genes of Drechmeria coniospora, Sci. Rep., 6, 23122. sets. Despite genetic predispositions for killing nematode, the micro- 11. Lebrigand, K., He, L.D., Thakur, N., et al. 2016, Comparative genomic scopic mechanism was different in NP fungi. Our analysis elucidated analysis of Drechmeria coniospora reveals core and speciﬁc genetic require- a repertoire of genes in endoparasitic NP fungi distinct from those of ments for fungal endoparasitism of nematodes, PLoS Genetics, 12,1–41. other NP fungi. The availability of the ﬁrst Ev genome sequencing, 12. Larriba, E., Jaime, M.D.L.A.M.D.L.A., Carbonell-Caballero, J.J., et al. 2014, annotation and evolution analysis was an important advance in help- Sequencing and functional analysis of the genome of a nematode egg-parasitic ing to decipher its pathogenesis. The results would facilitate our com- fungus, Pochonia chlamydosporia, Fungal Genet. Biol., 65, 69–80. prehension of the genomic features of NP fungi for adaptation to 13. Aranda-Martinez, A., Lenfant, N., Escudero, N., Zavala-Gonzalez, E.A., nematodes. Furthermore, the results also set out a solid theoretical Henrissat, B. and Lopez-Llorca, L.V. 2016, CAZyme content of pochonia chlamydosporia reﬂects that chitin and chitosan modiﬁcation are involved foundation for further research and application of these NP fungi as in nematode parasitism, Environ. Microbiol., 18, 4200–15. biological control agents. 14. Lai, Y., Liu, K., Zhang, X.X., et al. 2014, Comparative genomics and transcriptomics analyses reveal divergent lifestyle features of nematode endo- parasitic fungus Hirsutella minnesotensis, Genome Biol. Evol., 6, 3077–93. Acknowledgements 15. Liu, K., Zhang, W., Lai, Y., et al. 2014, Drechslerella stenobrocha ge- The authors would like to thank Chengshu Wang (Shanghai Institutes for nome illustrates the mechanism of constricting rings and the origin of Biological Sciences, CAS) and reviewers for suggestion for the manuscript modi- nematode predation in fungi, BMC Genomics, 15, 114. ﬁcation. This work was supported by the Fundamental Research Funds for the 16. Chu, W.H., Dou, Q., Chu, H.L., Wang, H.H., Sung, C K. and Wang, Central Non-proﬁt Research Institute of CAF (No. CAFYBB2017SZ003) of C.Y. 2015, Research advance on Esteya vermicola, a high potential bio- China, Institute Special Fund for Basic Research, Institute of Forest Ecology, control agent of pine wilt disease, Mycol. Progress, 14, 115. Environment, and Protection, Chinese Academy of Forestry (Grant no. 17. Chin, C.-S., Alexander, D.H., Marks, P., et al. 2013, Nonhybrid, ﬁnished CAFRIFEEP201403) and State 863 Project funded by Ministry of Science and microbial genome assemblies from long-read SMRT sequencing data, Technology of the People’s Republic of China (Grant no. 2012AA101503). Nat. Methods, 10, 563–9. 18. Stanke, M., Scho ¨ ffmann, O., Morgenstern, B. and Waack, S. 2006, Gene prediction in eukaryotes with a generalized hidden Markov model that Accession number uses hints from external sources, BMC Bioinformatics, 7, 62. 19. Besemer, J. and Borodovsky, M. 2005, GeneMark: web software for gene SRP097009 ﬁnding in prokaryotes, eukaryotes and viruses, Nucleic Acids Res., 33, W451. 20. Birney, E., Clamp, M. and Durbin, R. 2004, Genewise and Genomewise, Genome Res., 14, 988–95. Conflict of interest 21. Haas, B.J., Salzberg, S.L., Zhu, W., et al. 2008, Automated eukaryotic The authors declare no conﬂict of interest. gene structure annotation using EVidenceModeler and the program to as- semble spliced alignments, Genome Biol., 9, R7. 22. Zhang, J. and Madden, T.L. 1997, PowerBLAST: a new network BLAST application for interactive or automated sequence analysis and annota- Supplementary data tion, Genome Res., 7, 649–56. Supplementary data are available at DNARES online. 23. Moriya, Y., Itoh, M., Okuda, S., Yoshizawa, A.C. and Kanehisa, M. 2007, KAAS: an automatic genome annotation and pathway reconstruc- tion server, Nucleic Acids Res., 35, W182. References 24. Quevillon, E., Silventoinen, V., Pillai, S., et al. 2005, InterProScan: protein domains identiﬁer, Nucleic Acids Res., 33, W116. 1. Futai, K. 2013, Pine wood nematode, Bursaphelenchus xylophilus, Annu. 25. Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P.M. and Rev. Phytopathol., 51, 61–83. Henrissat, B. 2014, The carbohydrate-active enzymes database (CAZy) in 2. Vicente, C., Espada, M., Vieira, P. and Mota, M. 2012, Pine wilt disease: 2013, Nucl. Acids Res., 42, D490. a threat to European forestry, Eur. J. Plant Pathol., 133, 89–99. 26. Finn, R.D., Mistry, J., Schuster-Bo ¨ ckler, B., et al. 2006, Pfam: clans, web 3. Liou, J., Shih, J. and Tzean, S. 1999, Esteya, a new nematophagous genus tools and services, Nucleic Acids Res., 34, D247–51. from Taiwan, attacking the pinewood nematode (Bursaphelenchus xylo- 27. Rawlings, N.D., Barrett, A.J. and Finn, R. 2016, Twenty years of the philus), Mycol. Res., 103, 242–8. MEROPS database of proteolytic enzymes, their substrates and inhibitors, 4. Lin, F.,Ye, J.,Wang, H.,Zhang,A., Zhao,B.and Hendricks, M. 2013,Host Nucleic Acids Res., 44, D343–50. deception: predaceous fungus, Esteya vermicola, entices pine wood nematode 28. Saier, M.H., Reddy, V.S., Tsu, B.V., Ahmed, M.S., Li, C. and Moreno- by mimicking the scent of pine tree for nutrient, PLoS One, 8, e71676. Hagelsieb, G. 2016, The Transporter Classiﬁcation Database (TCDB): re- 5. Wang, C.Y., Fang, Z.M. and Wang, Z. 2011, Biological control of the cent advances, Nucleic Acids Res., 44, D372–9. pinewood nematode Bursaphelenchus xylophilus by application of the en- 29. Urban, M., Irvine, A.G., Cuzick, A. and Hammond-Kosack, K.E. 2015, doparasitic fungus Esteya vermicola, BioControl, 56, 91–100. Using the pathogen-host interactions database (PHI-base) to investigate 6. Wang, C.Y., Fang, Z.M., Sun, B.S., Gu, L. J., Zhang, K.Q. and Sung, plant pathogen genomes and genes implicated in virulence, Front. Plant C.K. 2008, High infectivity of an endoparasitic fungus strain, Esteya ver- Sci., 6, 605. micola, against nematodes, J. Microbiol., 46, 380–9. Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/3/245/4791394 by Ed 'DeepDyve' Gillespie user on 26 June 2018 R. Wang et al. 255 30. Lu, T., Yao, B. and Zhang, C. 2012, DFVF: database of fungal virulence 52. Lieberman, T.D., Michel, J.-B., Aingaran, M., et al. 2011, Parallel bacte- factors, Database (Oxford), 2012, bas032. rial evolution within multiple patients identiﬁes candidate pathogenicity 31. Liu, B. and Pop, M. 2009, ARDB–antibiotic resistance genes database, genes, Nat. Genet., 43, 1275–80. Nucleic Acids Res., 37, D443–7. 53. Trivedi, P. and Wang, N. 2014, Host immune responses accelerate patho- 32. Weber, T., Blin, K., Duddela, S., et al. 2015, AntiSMASH 3.0-A compre- gen evolution, ISME J., 8, 727–31. hensive resource for the genome mining of biosynthetic gene clusters, 54. Staats, M., van Baarlen, P., Schouten, A., van Kan, J.A.L. and Bakker, Nucleic Acids Res., 43, W237–43. F.T. 2007, Positive selection in phytotoxic protein-encoding genes of 33. R Development Core, T. 2012, R: a language and environment for statisti- Botrytis species, Fungal Genet. Biol., 44, 52–63. cal computing. R Foundation for Statistical Computing, Vienna, Austria. 55. Biswas, S. and Akey, J.M. 2006, Genomic insights into positive selection, ISBN 3-900051-07-0, http://www.R-project.org/ (15 March 2012, date Trends Genet., 22, 437–46. last accessed). 56. Teichert, I. and Nowrousian, M. 2011, Evolution of genes for secondary 34. Li, L., Stoeckert, C.J. and Roos, D.S. 2003, OrthoMCL: identiﬁcation of metabolism in fungi, Mycota XVI, 231–55. ortholog groups for eukaryotic genomes, Genome Res., 13, 2178–89. 57. Xu, Y., Orozco, R., Wijeratne, E.M.K., Gunatilaka, A.A.L., Stock, S.P. 35. Katoh, K. and Standley, D.M. 2013, MAFFT multiple sequence alignment and Molna ´ r, I. 2008, Biosynthesis of the cyclooligomer depsipeptide beau- software version 7: Improvements in performance and usability, Mol. vericin, a virulence factor of the entomopathogenic fungus Beauveria Biol. Evol., 30, 772–80. bassiana, Chem. Biol., 15, 898–907. 36. Castresana, J. 2000, Selection of conserved blocks from multiple align- 58. Xu, Y., Orozco, R., Kithsiri Wijeratne, E.M., et al. 2009, Biosynthesis of ments for their use in phylogenetic analysis, Mol. Biol. Evol., 17, 540–52. the cyclooligomer depsipeptide bassianolide, an insecticidal virulence fac- 37. Stamatakis, A. 2006, RAxML-VI-HPC: maximum likelihood-based phy- tor of Beauveria bassiana, Fungal Genet. Biol., 46, 353–64. logenetic analyses with thousands of taxa and mixed models, 59. Wang, B., Kang, Q., Lu, Y., Bai, L. and Wang, C. 2012, Unveiling the bio- Bioinformatics, 22, 2688–90. synthetic puzzle of destruxins in Metarhizium species, Proc. Natl. Acad. 38. Yang, Z. 2007, PAML 4: phylogenetic analysis by maximum likelihood, Sci. USA, 109, 1287–92. Mol. Biol. Evol., 24, 1586–91. 60. Li, G., Zhang, K., Xu, J., Dong, J. and Liu, Y. 2007, Nematicidal sub- 39. Hedges, S.B., Dudley, J. and Kumar, S. 2006, TimeTree: a public stances from fungi, Recent Pat. Biotechnol., 1, 212–33. knowledge-base of divergence times among organisms, Bioinformatics, 61. Liang, L., Wu, H., Liu, Z., et al. 2013, Proteomic and transcriptional analyses 22, 2971–2. of Arthrobotrys oligospora cell wall related proteins reveal complexity of fun- 40. De Bie, T., Cristianini, N., Demuth, J.P. and Hahn, M.W. 2006, CAFE: a gal virulence against nematodes, Appl. Microbiol. Biotechnol., 97, 8683–92. computational tool for the study of gene family evolution, Bioinformatics, 62. Andersson, K.-M., Kumar, D., Bentzer, J., Friman, E., Ahre ´ n, D. and 22, 1269–71. Tunlid, A. 2014, Interspeciﬁc and host-related gene expression patterns in 41. Letunic, I. and Bork, P. 2007, Interactive Tree Of Life (iTOL): an online nematode-trapping fungi, BMC Genomics,, 15, 968. tool for phylogenetic tree display and annotation, Bioinformatics, 23, 63. Li, J., Yu, L., Yang, J., et al. 2010, New insights into the evolution of 127–8. subtilisin-like serine protease genes in Pezizomycotina, BMC Evol. Biol., 42. Yang, J., Huang, X., Tian, B., Wang, M., Niu, Q. and Zhang, K. 2005, 10, 68. Isolation and characterization of a serine protease from the nematopha- 64. Zou, C.G., Tao, N., Liu, W.J., et al. 2010, Regulation of subtilisin-like gous fungus, Lecanicillium psalliotae, displaying nematicidal activity, protease prC expression by nematode cuticle in the nematophagous fun- Biotechnol. Lett., 27, 1123–8. gus Clonostachys rosea, Environ. Microbiol., 12, 3243–52. 43. Wang, B., Wu, W. and Liu, X. 2007, Puriﬁcation and characterization of 65. Yang, J., Zhao, X., Liang, L., et al. 2011, Overexpression of a a neutral serine protease with nematicidal activity from Hirsutella rhossi- cuticle-degrading protease Ver112 increases the nematicidal activity of liensis, Mycopathologia, 163, 169–76. Paecilomyces lilacinus, Appl. Microbiol. Biotechnol., 89, 1895–903. 44. Wang, B., Liu, X., Wu, W., Liu, X. and Li, S. 2009, Puriﬁcation, charac- 66. Langfelder, K., Streibel, M., Jahn, B., Haase, G. and Brakhage, A.A. terization, and gene cloning of an alkaline serine protease from a highly 2003, Biosynthesis of fungal melanins and their importance for human virulent strain of the nematode-endoparasitic fungus Hirsutella rhossilien- pathogenic fungi, Fungal Genet. Biol., 38, 143–58. sis, Microbiol. Res., 164, 665–73. 67. St Leger, R.J., Frank, D.C., Roberts, D.W. and Staples, R. C. 1992, 45. Boldo, J.T., Junges, A., do Amaral, K.B., Staats, C.C., Vainstein, M.H. Molecular cloning and regulatory analysis of the cuticle-degrading-protease and Schrank, A. 2009, Endochitinase CHI2 of the biocontrol fungus structural gene from the entomopathogenic fungus Metarhizium anisopliae, Metarhizium anisopliae affects its virulence toward the cotton stainer bug Eur. J. Biochem., 204,991–1001. Dysdercus peruvianus, Curr. Genet., 55, 551–60. 68. Małagocka, J., Grell, M.N., Lange, L., Eilenberg, J. and Jensen, A.B. 46. Tikhonov, V.E., Lopez-Llorca, L.V., Salinas, J. and Jansson, H.-B. 2002, 2015, Transcriptome of an entomophthoralean fungus (Pandora formi- Puriﬁcation and characterization of chitinases from the nematophagous cae) shows molecular machinery adjusted for successful host exploitation fungi Verticillium chlamydosporium and V. suchlasporium, Fungal and transmission, J. Invertebr. Pathol., 128, 47–56. Genet. Biol., 35, 67–78. 69. Ward, E., Kerry, B.R., Manzanilla-Lo ´ pez, R.H., et al. 2012, The 47. Collemare, J. and Lebrun, M.H. 2011, Fungal secondary metabolites: an- Pochonia chlamydosporia serine protease gene vcp1 is subject to regula- cient toxins and novel effectors in plant-microbe interactions, In: Martin, tion by carbon, nitrogen and pH: implications for nematode biocontrol, F. and Kamoun, S. (eds), Effectors in Plant-Microbe Interactions. John PLoS One, 7, e35657. Wiley & Sons, pp. 377–400. 70. Yang, J., Tian, B., Liang, L. and Zhang, K.Q. 2007, Extracellular enzymes 48. Wolpert, T.J., Dunkle, L.D. and Ciuffetti, L.M. 2002, Host-selective tox- and the pathogenesis of nematophagous fungi, Appl. Microbiol. ins and avirulence determinants: what’s in a name? Annu. Rev. Biotechnol., 75, 21–31. Phytopathol., 40, 251–85. 71. Prasad, P., Varshney, D. and Adholeya, A. 2015, Whole genome annota- 49. Dean, R.A., Talbot, N.J., Ebbole, D.J., et al. 2005, The genome sequence tion and comparative genomic analyses of bio-control fungus of the rice blast fungus Magnaporthe grisea, Nature, 434, 980–6. Purpureocillium lilacinum, BMC Genomics, 16, 1004. 50. Powell, A.J., Conant, G.C., Brown, D.E., Carbone, I. and Dean, R.A. 72. Wanyiri, J.W., Techasintana, P., O’Connor, R.M., Blackman, M.J., Kim, 2008, Altered patterns of gene duplication and differential gene gain and K. and Ward, H.D. 2009, Role of CpSUB1, a subtilisin-like protease, in loss in fungal pathogens, BMC Genomics, 9, 147. cryptosporidium parvum infection in vitro, Eukaryotic Cell., 8, 470–7. 51. Fillinger, S., Chaveroche, M.K., van Dijck, P., et al. 2001, Trehalose is 73. Martinez, D., Challacombe, J. and Morgenstern, I. 2009, Genome, tran- required for the acquisition of tolerance to a variety of stresses in scriptome, and secretome analysis of wood decay fungus Postia placenta the ﬁlamentous fungus Aspergillus nidulans, Microbiology, 147, supports unique mechanisms of lignocellulose conversion, Proc. Natl. 1851–62. Acad. Sci. USA, 106, 1954–9. Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/3/245/4791394 by Ed 'DeepDyve' Gillespie user on 26 June 2018 256 Genome features of nematophagous fungi 74. Schuler, D., Wahl, R., Wippel, K., et al. 2015, Hxt1, a monosaccharide 82. de la Cruz, J., Pintor-Toro, J.A., Benı´tez, T., Llobell, A. and Romero, L.C. transporter and sensor required for virulence of the maize pathogen 1995, A Novel endo-beta-1,3-glucanase, BGN13.1, involved in Ustilago maydis, New Phytol., 206, 1086–100. the mycoparasitism of Trichoderma harzianum, J. Bacteriol., 177, 75. Cui, Z., Gao, N., Wang, Q., Ren, Y., Wang, K. and Zhu, T. 2015, 6937–45. BcMctA, a putative monocarboxylate transporter, is required for pathoge- 83. El-Katatny, M.H., Gudelj, M., Robra, K.H., Elnaghy, M.A. and Gu ¨ bitz, nicity in Botrytis cinerea, Curr. Genet., 61, 545–53. G.M. 2001, Characterization of a chitinase and an endo-b-1, 3-glucanase 76. Doherty, C.P. 2007, Host-pathogen interactions : the Role of Iron, from Trichoderma harzianum Rifai T24 involved in control of the J. Nutr., 137, 1341–4. phytopathogen Sclerotium rolfsii, Appl. Microbiol. Biotechnol., 56, 77. Haas, H., Eisendle, M. and Turgeon, B.G. 2008, Siderophores in fungal 137–43. physiology and virulence, Annu. Rev. Phytopathol., 46, 149–87. 84. Zhao, L.L., Wei, W., Kang, L. and Sun, J.H. 2007, Chemotaxis of the 78. Haas, H. 2012, Iron—a key nexus in the virulence of Aspergillus fumiga- pinewood nematode, Bursaphelenchus xylophilus, to volatiles associated tus, Front. Microbiol., 3, 1–10. with host pine, Pinus massoniana, and its vector Monochamus alternatus, 79. Ibrahim, A.S., Gebremariam, T., Lin, L., et al. 2010, The high afﬁnity iron J. Chem. Ecol., 33, 1207–16. permease is a key virulence factor required for Rhizopus oryzae pathogen- 85. Niu, H., Zhao, L., Lu, M., Zhang, S., Sun, J. and Yang, C.-H. 2012, The esis, Mol. Microbiol., 77, 587–604. ratio and concentration of two monoterpenes mediate fecundity of the 80. Berg, R.H., Fester, T. and Taylor, C.G. 2009, Cell Biology of Plant pinewood nematode and growth of its associated fungi, PLoS One, 7, Nematode Parasitism. In: Berg R.H. and Taylor C.G. (eds), Plant cell e31716. monographs, Vol. 15. Springer-Verlag: Berlin, pp. 115–52. 86. Wang, R., Dong, L., Chen, Y., Qu, L., Wang, Q. and Zhang, Y. 2017, 81. Pitson, S., Seviour, R.J., Bott, J. and Stasinopoulos, S.J. 1991, Production Esteya Vermicola, a nematophagous fungus attacking the pine wood nem- and regulation of b-glucanases in Acremonium and Cephalosporium iso- atode, harbors a bacterial endosymbiont afﬁliated with gammaproteobac- lates, Mycol. Res., 95, 352–6. teria, Microbes Environ., 32, 201–9. Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/3/245/4791394 by Ed 'DeepDyve' Gillespie user on 26 June 2018
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