Distinctive Expansion of Potential Virulence Genes in the Genome of the Oomycete Fish Pathogen Saprolegnia parasitica
[['Saprolegnia species are watermolds or oomycetes that are endemic to probably all fresh water ecosystems .', 'These understudied pathogens can cause destructive diseases of amphibians , crustaceans , fish and insects in aquaculture and in natural environments worldwide [1] , [2] .', 'With the rise of fish as a principal source of animal protein , and the decline of wild fish stocks , aquaculture production has increased on average by 11% per year worldwide over the past ten years ( FAO Fishery Information ) .', 'Intensive aquatic farming practices have resulted in explosive growth in pathogen populations , which has been exacerbated by the ban of malachite green as a pesticide .', 'Losses due to microbial , parasitic and viral infections are the largest problem in fish farms nowadays , and have a significant effect on animal welfare and sustainability of the industry .', 'The salmon farming industry is particularly affected by Saprolegnia parasitica .', 'This pathogen causes Saprolegniosis ( also known as Saprolegniasis ) , a disease characterized by visible grey or white patches of mycelium on skin and fins , and subsequent penetration of mycelium into muscles and blood vessels [1] , [3] .', 'It is estimated that 10% of all hatched salmon succumb to Saprolegnia infections and losses are estimated at tens of millions of dollars annually [2] .', 'In addition to the damage to the aquaculture industry , the declines of natural salmonid populations have also been attributed to Saprolegnia infections [1] .', 'More in-depth knowledge of the epidemiology , biology and pathology of the pathogen is urgently needed .', 'A draft genome sequence of S . parasitica provides an excellent starting point to investigate the disease process at the molecular and cellular level and may lead to novel avenues for sustainable control of Saprolegniosis .', 'Animal pathogens have evolved independently multiple times in lineages such as Stramenopila , Alveolata , Amebozoa , Euglenozoa and Mycota , as well as in numerous bacterial lineages .', 'Oomycetes such as Saprolegnia belong to the kingdom Stramenopila ( Patterson , 1989 ) , syn .', 'Straminipila ( Dick , 2001 ) , that includes photosynthetic algae such as kelp and diatoms , ubiquitous saprotrophic flagellates such as Cafeteria roenbergensis , and obligate mammalian parasites such as Blastocystis [4] , [5] .', 'Although many Saprolegnia and related species are capable of causing diseases on a wide range of animal hosts including humans , relatively little is known about their mechanisms of pathogenicity .', 'Among the oomycetes , most animal pathogens including S . parasitica belong to the class Saprolegniomycetidae ( Figure 1A ) .', 'The oomycetes also include many plant pathogens and these are mainly concentrated within the class Peronosporomycetidae .', 'There are a small number of interesting exceptions to this otherwise sharp dichotomy , including the mammalian pathogen Pythium insidiosum ( Peronosporomycetidae ) and the plant pathogens Aphanomyces euteiches and Aphanomyces cochlioides ( Saprolegniomycetidae ) [1] , [6] , [7] .', 'Plant pathogens in the class Peronosporomycetidae include Phytophthora and Pythium species , downy mildew pathogens and white rusts ( Figure 1A ) .', 'Well-known examples are the potato late blight pathogen Phytophthora infestans that caused the great Irish famine in the 1840s [8] , the soybean root rot pathogen Phytophthora sojae [9] , and the sudden oak death pathogen Phytophthora ramorum [10] .', 'Sequenced and assembled genomes have been generated for P . sojae and P . ramorum [11] , P . infestans [12] , and for several relatives including the broad host range plant pathogens Phytophthora capsici [13] and Pythium ultimum [14] , the downy mildew pathogen of Arabidopsis , Hyaloperonospora arabidopsidis [15] , and the white blister rust of Brassicaceae , Albugo candida [16] .', 'Genome analyses have revealed a bi-partite genome organization in these pathogens , in which gene dense regions containing clusters of orthologs with well-conserved sequences and gene order are interspersed with repeat-rich regions containing rapidly evolving families of virulence genes and numerous transposons [12] , [17] , [18] .', 'The virulence gene families of plant pathogenic oomycetes encode numerous hydrolytic enzymes for degradation of plant carbohydrates , extracellular toxins such as NLP and PcF toxins , and at least three families of cell-entering effector proteins , RXLR effectors , CHXC effectors and Crinkler proteins [7] , [11] , [19] , [20] .', 'These classes of effector proteins contain amino acid sequence motifs ( RXLR , CHXC , and LFLAK respectively ) that are involved in entry into the host plant cell [12] , [21] , [22] , [23] , [24] , [25] and effector domains that target diverse host physiological processes to suppress immunity and promote infection [17] .', 'In the genome of the broad host-range necrotrophic oomycete Py .', 'ultimum , which primarily targets immature or stressed plant tissues , enzyme families are expanded that enable degradation of readily accessible carbohydrates such as pectins , starch , and sucrose , while RXLR effectors appear to be completely absent [14] .', 'In the obligate biotroph H . arabidopsidis , most gene families are smaller , especially those encoding hydrolytic enzymes [15] .', 'Interestingly , EST libraries from the saprolegniomycete plant pathogen A . euteiches revealed the presence of Crinkler effectors ( but neither NLP toxins nor RXLR effectors ) [26] , suggesting that Crinklers are ancestral to oomycete pathogens [23] .', 'So far , only limited genomic resources are available for animal pathogenic oomycetes .', 'Analysis of small sets of EST data of S . parasitica [27] , [28] and Py .', 'insidiosum [29] , revealed the presence of secreted protein families with potential roles in virulence such as glycosyl hydrolases , proteases , and protease inhibitors , as well as proteins involved in protection against oxidative stress .', 'The S . parasitica data set included a host-targeting protein SpHtp1 ( S . parasitica host-targeting protein 1 ) that was subsequently demonstrated to enter fish cells through binding to a tyrosine-O-sulfated fish cell surface ligand [30] .', 'Here we report the genome sequence and transcriptome analysis of S . parasitica , the first genome sequenced from an animal pathogenic oomycete .', 'We compared the genome to those of plant pathogenic oomycetes , revealing distinctive genome expansions and adaptations tailored to the physiology of the respective hosts .', 'This study adds to our understanding of mechanisms for invasion and colonization of animal host cells by eukaryotic pathogens .'], ['The strategy of whole genome shotgun sequencing was applied to S . parasitica strain CBS223 . 65 , which is a strain isolated from infected pike ( Esox lucius ) .', 'A combination of 454 ( fragment and 3 kb jumping libraries ) and Sanger ( Fosmid library ) sequencing data ( ∼50-fold average read coverage ) were used to assemble the genome , yielding an initial assembly length of 53 Mb and a scaffold N50 length of 281 kb ( see Table S1 for additional assembly statistics ) .', 'Read coverage and rates of polymorphism were computed based on alignments of Illumina data ( generated for polymorphism discovery ) to the genome assembly .', 'Based on the distribution of coverage and polymorphisms ( Figure S1A and S1B ) , we conclude that the assembly represents a composition of regions of diploid consensus ( 76% of the assembly ) with the remainder corresponding to separately assembled haplotypes , resulting from the high polymorphism rate with a peak of 2 . 6% ( Table S2 and Figure S1C ) .', 'Taking into account the 24% of the assembly corresponding to separately-assembled haplotypes , the total assembled haploid genome size was adjusted to 42 . 3 Mb ( see Text S1 ) .', 'Based on read coverage we estimated the total genome size to be 62 . 8 Mb .', 'The remaining 20 . 4 Mb of genomic sequence were estimated to correspond to collapsed tandem repeat content in the genome assembly ( Text S1 ) .', 'Read coverage analysis of the separated haplotypes and of regions showing loss of heterozygosity ( see below ) produced an overall genome size estimate of 63 Mb ( Text S1 ) , consistent with our effective assembled genome size estimate .', 'The difference between the assembly size and the read coverage estimate likely results from tandem repeats collapsed in the assembly and uncertainties in read alignments , as observed in other oomycete genome sequence assemblies ( Table 1 ) .', 'More than 98% of Trinity [31] de novo assembled transcripts from in vitro growth RNA-Seq data ( described in Material and Methods ) mapped to the genome assembly , indicating that the expressed gene content is very well represented by the assembled genome .', 'The genome size of S . parasitica is consistent with the size range of most previously sequenced oomycete genomes , which rank from 45 Mb ( Py . ultimum ) [14] to 65 Mb ( P . ramorum ) [11] , far below the outlier of 240 Mb ( P . infestans ) [12] ( Table 1 ) .', 'Approximately one-third of the assembled S . parasitica genome was found to correspond to regions exhibiting loss of heterozygosity ( LOH ) ( Figure S1C and Text S1 ) .', 'LOH resulting from mitotic instability has been observed in other oomycete genomes [13] , [32] , [33] , and provides a potential adaptive mechanism that promotes expression of genetic diversity within a clonal pathogen population [13] .', 'The prevalence of LOH within a S . parasitica population and the role LOH plays in S . parasitica evolution remain to be determined .', 'A total of 20 , 113 coding gene models were computationally predicted , with 3 , 048 pairs of coding genes assigned as alleles within separately assembled haplotype regions ( Table S3 ) , yielding an adjusted coding gene count of 17 , 065 , similar in magnitude to counts of genes identified in other sequenced oomycete genomes ( Table 1 ) .', 'There are 5 , 291 coding genes ( 31% of total ) found to reside within regions of LOH ( Table S3 ) .', 'No obvious enrichment or depletion of biologically relevant gene functions could be detected within the defined regions of LOH that would suggest that the LOH observed had resulted from selection in the individual strain sequenced .', 'The gene density found in S . parasitica is one of the highest reported so far for oomycetes , with one gene per 2 . 6 kb ( Table 1 ) .', 'This is slightly denser than in Py .', 'ultimum ( one gene per 2 . 9 kb ) and A . candida ( 2 . 9 kb ) , and much denser than in P . infestans ( one gene per 10 . 7 kb ) .', 'Many of the S . parasitica genes are novel; only 40% of the S . parasitica predicted proteins have homologs with more than 50% sequence similarity to those from other organisms , including oomycetes .', 'A conserved core-proteome of 4 , 215 proteins can be identified for plant pathogenic peronosporomycetes based on genomes from multiple Phytophthora species , H . arabidopsidis and Py .', 'ultimum [14] .', 'Of these 4215 , S . parasitica shares only 3518 orthologs ( Figure S2A ) , of which only about 40% show strong sequence similarity ( >50% ) .', '20% of the core set is not detectable in the S . parasitica proteome ( Figure S2B ) .', 'Although the genomes of the peronosporomycetes show substantial conservation of gene order ( synteny ) , little of this synteny is preserved in S . parasitica , as was observed for A . candida [16] .', 'Interestingly , compared to other oomycetes , S . parasitica genes contain a larger number of introns ( Figure 1B , Table 1 ) .', 'More than 73% of the S . parasitica genes contain at least one intron , compared to 50–60% in other oomycete species ( Table 1 ) .', 'Among 4008 orthologs shared between S . parasitica and three Phytophthora species , the majority of the genes have different numbers of introns .', 'For example , more than half of the S . parasitica genes have more introns than their orthologs in Phytophthora , and 15% of the S . parasitica genes have 5 or more additional exons compared to their Phytophthora orthologs ( Figure 1C ) .', 'The intron abundance in S . parasitica potentially more closely matches the ancestral state , assuming a trend of intron reduction as found in animal and fungal lineages [34] .', 'The S . parasitica genome has very few known mobile elements , which is consistent with its smaller size compared to the transposon-rich Phytophthora genomes .', 'Of the 160 repeat families identified among all sequenced Phytophthora species , only one LTR retrotransposon family was found in the S . parasitica genome ( Figure S3A ) .', 'This group of LTR elements , which occur at low copy numbers ( <20 ) in known oomycete genomes ( Figure S3B ) , is thus ancient .', 'The largest transposon family in S . parasitica ( approx . 50 copies in the assembled sequence , and an estimated a few hundred copies in the genome ) belongs to the LINE repeat group ( Text S1 ) .', 'LINE elements are abundant in animal genomes and play roles in genome evolution and modulation of gene expression [35] .', 'Curiously , the S . parasitica LINE element family is absent from the Phytophthora genomes but shares sequence similarity with the LINE elements from animal genomes ( Figure S3C ) , raising the possibility that this family was acquired from an animal host .', 'Eukaryotic protein kinases ( ePKs ) regulate a myriad of cellular activities by phosphorylating target proteins in response to internal or external signals .', 'S . parasitica has one of the largest kinomes that have been identified to date , with 543 predicted ePKs .', 'For example , S . parasitica has 65 more ePKs than the human kinome by the same prediction criteria ( Figure S4A ) .', 'Eukaryotic protein kinases have been classified into eight major groups based on sequence similarity .', 'We classified members of the S . parasitica ePK superfamily using a set of HMMs based on previously identified kinases ( Figure S4A ) .', 'S . parasitica has a large expansion of tyrosine kinase-like proteins with 298 members and a large number of unclassified kinases ( 114 ) , suggesting novel functions performed by the S . parasitica kinome .', 'Interestingly , the S . parasitica kinome contains several kinase families that have typically not been seen outside the metazoan clade , for example CAMK2 , NUAK , SNRK , and PHK from the Ca2+ calmodulin-dependent kinase group .', 'Some of these “metazoan” kinases are shared with plant pathogenic oomycetes [36] , substantially pushing back the evolutionary origin of these kinases .', 'We found 131 kinases containing predicted transmembrane helices ( Figure S4B ) , suggesting that S . parasitica has a large number of protein kinases that may function as cell surface receptors with roles in signaling .', 'Such a large receptor repertoire may facilitate the recognition of stimuli from extracellular abiotic and biotic environments .', 'The cell wall of a pathogen plays a central role at the host-pathogen interface .', 'In particular , cell wall related proteins and polysaccharides are a large source of PAMPs ( Pathogen Associated Molecular Patterns ) of both animal and plant pathogens [37] , [38] .', 'In addition , cell wall carbohydrate biosynthetic enzymes represent a potential target of antimicrobial compounds when similar activities are not encountered in the host .', 'This is illustrated by the demonstration that the specific inhibition of chitin synthase ( CHS ) in Saprolegnia leads to cell death although chitin represents no more than 1% of the total cell wall carbohydrates of the pathogen [39] , [40] .', 'Chitin is a structural crystalline polymer typically associated with the fungal cell wall .', 'Historically , the absence of chitin in Phytophthora [41] has led to the general concept that oomycetes are devoid of chitin and that cellulose , the major load-bearing structural polysaccharide in oomycete walls , is a key feature distinguishing oomycetes from true fungi .', 'However , the occurrence of chitin has since been demonstrated in various oomycete species belonging to the Saprolegniales [39] , [40] , [42] .', 'In addition , GlcNAc-based carbohydrates that do not seem to correspond to crystalline chitin , but whose biosynthesis is most likely performed by putative chitin synthase gene products are present in the walls of Aphanomyces euteiches [43] .', 'The S . parasitica genome appears to contain genes that encode enzymes involved in chitin biosynthesis , modification and degradation ( Table S4 ) .', 'Out of these , six genes correspond to putative chitin synthases ( SPRG_02074 , SPRG_02554 , SPRG_04151 , SPRG_09812 , SPRG_06131 and SPRG_19383 ) .', 'Interestingly , this number is higher than in other oomycetes where only one or two CHS putative genes have been detected [39] , [43] ( Table S4 ) .', 'The only oomycete CHS gene product for which the function has been unequivocally demonstrated through heterologous expression and in vitro biochemical assays is CHS2 from Saprolegnia monoica [39] .', 'Thus , as for most CHS genes from other oomycetes , the function of the S . parasitica genes annotated here as CHS remains to be demonstrated .', 'As reported earlier in S . monoica [39] , two of the newly identified S . parasitica CHS gene products ( SPRG_09812 and SPRG_04151 ) contain a so-called MIT ( Microtubule Interacting and Trafficking ( or Transport ) ) domain .', 'These MIT domains are possibly involved in the trafficking and delivery of the corresponding CHS at the apex of the hyphal cells , as previously suggested for S . monoica [39] .', 'There are also a large number ( 14 ) of chitinase genes in S . parasitica compared to other oomycetes , which could represent many ancestral forms of oomycete chitinases ( Figure 1D ) .', 'There are major structural and physiological differences between plant and animal cells , and thus the metabolisms of plant and animal pathogens have likely adapted accordingly to the respective host cellular environments .', 'Pectin is a major constituent of plant cell walls and a target for extracellular enzymes produced by pathogenic and saprophytic microorganisms .', 'Plant pathogenic fungi and oomycetes produce a large array of enzymes to degrade pectin , including polygalacturonase ( PG ) , pectin and pectate lyases ( PL ) , and pectin methylesterases ( PME ) .', 'Animal cells lack a cell wall , and as might be expected , the pathogen S . parasitica encodes very few cell wall degrading enzymes in its genome .', 'Genes encoding hydrolytic enzymes such as cutinase and pectin methyl esterases appear to be absent , and PL and PG genes are greatly reduced in numbers as compared to plant pathogenic oomycetes ( Table 2 ) .', 'The remaining small numbers of PLs and PGs may play a role in the saprophytic life stage of S . parasitica in the aquatic environment outside of fish hosts .', 'S . parasitica proliferates in host tissue rich in proteins and ammonium .', 'Concomitantly , its pathways involved in inorganic nitrogen and sulfur assimilation have degenerated ( Figure 2A ) .', 'The loss of these metabolic capabilities has occurred independently in the obligate oomycete plant pathogen H . arabidopsidis [15] , as well as within several lineages of obligate fungal plant pathogens , presumably due to the high level of parasitic adaptation in these organisms .', 'Strikingly the same physical clusters of genes have been lost in each lineage , namely the genes encoding nitrate reductase , nitrite reductase , sulfite reductase and nitrate transporters ( Table S5 ) .', 'Also in line with a protein-rich environment that is a major source of both carbon and nitrogen , the S . parasitica genome contains 56 genes predicted to encode amino acid transporters .', 'Most of the S . parasitica transporters appear to be novel because less than 20 of the predicted amino acid transporters have orthologs or closely related paralogs in other oomycete genomes .', 'Phylogenetic analysis shows there are lineage-specific expansions of amino acid transporter genes in the different oomycete genomes , with recently duplicated S . parasitica genes forming the largest group ( Figure 2B ) .', 'The gene for phospholipase C ( PLC ) is absent in all of the sequenced peronosporomycete plant pathogens , but is present in S . parasitica ( SPRG_04373 ) .', 'Phylogenetic analysis groups the S . parasitica PLC gene with that of other heterokont species ( Figure S5 , Table S6 ) .', 'This shows that the S . parasitica PLC is most likely to be ancestral and that the absence of PLC in other oomycetes is due to gene loss .', 'Peronosporomycete plant pathogens are sterol auxotrophs and their genomes are missing most genes encoding enzymes involved in sterol biosynthesis [44] .', 'In contrast , analysis of the EST collection from A . euteiches and the S . parasitica genome predicts the existence of enzymes that function in a novel sterol biosynthetic pathway [26] which has been shown to lead to the synthesis of fucosterol in A . euteiches [45] .', 'Importantly , one of the genes SPRG_09493 encodes a CYP51 sterol-demethylase ( Figure S6 ) , a major target of antifungal chemicals that could perhaps also be used to combat Saprolegniomycetes .', 'Like plant pathogens , S . parasitica presumably secretes a battery of virulence proteins to promote infection .', 'Due to co-evolution with the host , virulence proteins are typically rapidly evolving and may appear to be unique to the species , or encoded by recently expanded gene families [17] , [19] .', 'The S . parasitica genome contains a large number of genes ( 11 , 825 ) that are not orthologous to any known genes in other species ( Figure S2A and S2B ) , and many recently expanded gene families .', 'There are at least 87 pfam domains that are either unique or show recent expansions in S . parasitica as compared to other oomycete species ( Table S7 ) .', 'An estimated 970 proteins ( Table S8 ) were predicted to be extracellular based on previously established bioinformatics criteria [11] , [12] , such as the presence of a eukaryotic signal peptide , and lack of targeting signals to organelles or membranes .', 'Many of the expanded families appear to function at the exterior or cell surface of the pathogens , such as proteins containing CBM1 ( Carbohydrate Binding Module Family I according to the CAZy database ( http://www . cazy . org/; [46] ) , ricin B lectin , Notch domains , and also numerous peptidases .', 'Among the proteins that are unique to S . parasitica compared to plant pathogenic oomycetes , the largest families have similarities to animal-pathogenesis-associated proteins , such as disintegrins , ricin-like galactose-binding lectins and bacterial toxin-like proteins ( haemolysin E ) .', 'Oomycetes contain an unusually large number of proteins with novel domain combinations , recruited from common metabolic , regulatory and signaling domains [47] , [48] .', 'S . parasitica contains in total 169 novel domain combinations that are specific to this pathogen ( Table S9 ) .', 'As described above , some of the lineage-expanded domains such as CBM and ricin are used for novel combinations to form composite proteins .', 'Additional domains used for novel combinations are the cytochrome p450 and tyrosinase domains .', 'Proteins carrying S . parasitica-specific domain combinations are significantly enriched ( hypergeometric test , p<0 . 001 ) in predicted secreted proteins ( 3 . 6% of secretome ) , whereas only 1 . 2% of total proteins have S . parasitica-specific novel combinations .', 'The enrichment in secreted proteins is strongly suggestive of a role for the novel domain combinations in pathogenesis .', 'There are about 1000 proteins that are predicted to be secreted by S . parasitica , based on criteria used for secretome prediction in other oomycetes [11] , [12] .', 'Two groups of proteins dominate the secretome of S . parasitica: proteases and lectins ( Figure 3A , Table S8 ) .', 'There are over a hundred members in the each of the two groups .', 'In the proteome , S . parasitica has one of the largest repertoires of proteases ( 270 ) known to date compared to most other single cell or filamentous eukaryotic pathogens ( such as most sequenced fungi species ) that typically have between 70 and 150 proteases .', 'In almost every family of proteases , including cysteine- , serine- and metallo-proteases ( Table 2 ) , there is an expansion in S . parasitica compared to P . sojae ( Figure 2C ) .', 'The most relatively abundant family of proteases is the papain-like peptidase_C1 proteins , comprising 48 proteins; the other oomycetes contain only about 20 proteins .', 'The majority of papain-like peptidase_C1 genes ( 80% ) have been generated by S . parasitica lineage-specific gene duplications and form a lineage-specific clade in the phylogenetic reconstruction ( Figure 2D ) .', 'Amongst the cell-surface associated proteins , ricin_B_lectin-like proteins and CBM1 domain-containing proteins are most abundant ( Table 2 ) .', 'There are 40 ricin-like and 40 CBM1 genes in S . parasitica , a large expansion compared to other known oomycetes .', 'In some cases , these domains are fused to other secreted protein domains having catalytic activities ( protease and cellulose ) to form novel proteins unique to oomycetes ( Figure 3B ) .', 'However , typical peronosporomycete proteins were not found , such as CBEL ( Cellulose Binding Elicitor Lectin ) , which contains a CBM1-PAN domain association and mediates the binding of mycelium to cellulosic substrates [49] .', 'The saprolegniomycete plant pathogen Aphanomyces euteiches also lacked CBEL proteins [26] .', 'We investigated whether culture filtrates containing secreted proteins from S . parasitica could degrade trout immunoglobulin M ( IgM ) as previously found for bacterial fish pathogens [50] .', 'Although no effect was observed when supernatants of two-day old cultures were incubated with an IgM enriched fraction ( data not shown ) , the 7-day post-inoculation supernatant degraded the IgM protein fraction within several hours ( Figure 4A ) .', 'No degradation of trout IgM was detected when heat-treated 7-day post-inoculation supernatant was used .', 'The protease inhibitors EDTA ( a metalloproteinase inhibitor ) and PMSF ( serine protease inhibitor ) showed partial inhibition of the IgM-degrading activity while E-64 ( a cysteine protease inhibitor ) did not show any inhibition .', 'The combination of EDTA and PMSF prevented IgM degradation and the detection was similar to the pea broth control .', 'These results suggest that secreted proteases from S . parasitica could degrade fish IgM and that metalloproteinases and serine proteases may be the classes involved in this process .', 'To further characterize the IgM-degrading properties of S . parasitica proteases , a serine protease ( SPRG_14567 ) was selected from the S . parasitica genome based on our observation that this protease possesses a secretory signal peptide ( Figure 4B ) and is highly expressed ( RNA-Seq data ) ( Figure 4C ) .', 'Interestingly , SPRG_14567 showed strong degrading activity towards trout IgM while no activity was detected when control E . coli soluble proteins were used ( Figure 4B and 4E ) .', 'This indicates that this serine protease is capable of degrading fish IgM and could be a virulence factor that combats the activity of fish immunoglobulins against Saprolegnia .', 'To establish a successful infection , pathogens often deliver effector proteins and toxins into the host cytoplasm to manipulate host immunity [7] , [24] , [51] .', 'Analogous to bacterial Type II secreted toxins that enter host cells via lipid-receptor-mediated endocytosis , plant pathogenic oomycetes utilize a host-targeting domain to deliver effectors into plant cells .', 'Hundreds of effectors carrying the host targeting motifs of RXLR and LFLAK have been identified in plant pathogenic oomycetes [11] , [12] , [22] .', 'However , these large families of Crinkler and RXLR effectors appear to be absent in S . parasitica ( Table 2 ) .', 'Using sensitive BLAST and HMM searches based on the RXLR domain and C-terminal domains of effectors , no plant pathogen-like RXLR effectors could be detected in the genome .', 'Bioinformatic searches with the de novo motif-finding program MEME did not identify other putative host-targeting motifs .', 'Despite the absence of large RXLR effector families , S . parasitica does have a small family of host targeting proteins related to SpHtp1 , which do contain an N-terminal RXLR sequence .', 'Interestingly one of these proteins was shown to translocate into fish cells [28] and entry required the N-terminal leader sequence [30] .', 'The lack of sequence similarity between any part of SpHtp1 and RXLR-proteins from plant pathogen oomycetes , except for the three proximally located amino acid residues , suggests that the presence of the RXLR-sequence in SpHtp1 is currently unclear .', 'No significant matches were found to Crinkler effectors , suggesting they are absent from S . parasitica .', 'Using search criteria that do not rely on sequence homology , such as induction of expression during the pre-infection stage , presence of secretion signals , and signatures of fast evolution , several candidates ( Table S10 ) for host-targeting proteins ( including SpHtp1 ) were identified in S . parasitica .', 'None of these candidates have homology to known proteins , and their functions are currently unknown .', 'The commonalities between animal and plant pathogenic oomycetes are highlighted by their shared PAMPs .', 'Proteinaceous PAMPs found in plant pathogenic oomycetes , namely CBM1 [37] , elicitins , and Cys-rich-family-3 proteins [14] are found in S . parasitica ( Figure S7A ) .', 'Elicitins are extracellular lipid transfer proteins that elicit defense responses in some species of plants , especially Nicotiana [52] .', 'There are 29 elicitin-like proteins in S . parasitica .', 'Phylogenetic reconstruction shows that the majority of the S . parasitica elicitins form three lineage-specific clades , distant from the canonical elicitin group [53] ( Figure S7B ) .', 'We also detected six YxSL[RK] containing secreted proteins , previously identified as candidate effectors [14] , that also show sequence divergence from known members of this family .', 'It is an intriguing question whether animal innate immune systems can detect these potential PAMPs , as does the plant innate immune system .', 'For example it seems unlikely that CBM1 can act as immune elicitor for animal cells since binding to the plant cell wall cellulose is required to induce immune responses in plants [37] .', 'To study the polymorphism and evolutionary rate of S . parasitica genes , we sequenced a related strain , VI-02736 , which was isolated from an infected Atlantic salmon , for comparison .', 'More than of 90% of the CBS223 . 65 reference sequence was covered by VI-02736 reads , allowing us to identify 1 , 467 , 567 SNPs between the two strains , giving an average SNP rate of 3 . 3% ( Text S1 , Table S11 and Figure S8A and S8B ) .', 'We have also determined that LOH is unique to the CBS strain , as it is apparently absent in the sequenced VI-02736 strain ( Text S1 , Figure S8C and S8D ) .', 'The Ka/Ks ratio ( Ka - number of non-synonymous substitutions per non-synonymous site , Ks - number of synonymous substitutions per synonymous site ) was calculated for the annotated S . parasitica gene set .', 'The set of 3518 oomycete core ortholog genes gives a median Ka/Ks ratio of 0 . 05 , a rate comparable to previously published Ka/Ks rate of the conserved gene dense region of Phytophthora sibling species and related strains [54] .', 'Non-parametric Z tests between the core ortholog group and a particular gene family ( listed in Table', '2 ) were performed to determine which family shows elevated substitution rates .', 'Among the pathogenesis-related gene families , we identified four groups , namely elicitins , disintegrins , host targeting proteins and haemolysins , that have significantly elevated median Ka/Ks ratios as compared to the core-ortholog groups ( Figure S9A ) .', 'Between the two S . parasitica strains , the highest Ka/Ks ratio was observed in the haemolysin E family , a group of toxin-like genes that were possibly horizontally acquired from bacteria ( see below ) .', 'Interestingly , similar patterns of elevated Ka/Ks ratios were also found between haplotypes of the reference strain ( Figure S9B ) .', 'Three families , the disintegrins , elicitins and haemolysins , show significant elevat
['Introduction', 'Results', 'Discussion', 'Methods']
['Oomycetes in the class Saprolegniomycetidae of the Eukaryotic kingdom Stramenopila have evolved as severe pathogens of amphibians , crustaceans , fish and insects , resulting in major losses in aquaculture and damage to aquatic ecosystems .', 'We have sequenced the 63 Mb genome of the fresh water fish pathogen , Saprolegnia parasitica .', 'Approximately 1/3 of the assembled genome exhibits loss of heterozygosity , indicating an efficient mechanism for revealing new variation .', 'Comparison of S . parasitica with plant pathogenic oomycetes suggests that during evolution the host cellular environment has driven distinct patterns of gene expansion and loss in the genomes of plant and animal pathogens .', 'S . parasitica possesses one of the largest repertoires of proteases ( 270 ) among eukaryotes that are deployed in waves at different points during infection as determined from RNA-Seq data .', 'In contrast , despite being capable of living saprotrophically , parasitism has led to loss of inorganic nitrogen and sulfur assimilation pathways , strikingly similar to losses in obligate plant pathogenic oomycetes and fungi .', "The large gene families that are hallmarks of plant pathogenic oomycetes such as Phytophthora appear to be lacking in S . parasitica , including those encoding RXLR effectors , Crinkler's , and Necrosis Inducing-Like Proteins ( NLP ) .", 'S . parasitica also has a very large kinome of 543 kinases , 10% of which is induced upon infection .', 'Moreover , S . parasitica encodes several genes typical of animals or animal-pathogens and lacking from other oomycetes , including disintegrins and galactose-binding lectins , whose expression and evolutionary origins implicate horizontal gene transfer in the evolution of animal pathogenesis in S . parasitica .']
['Fish are an increasingly important source of animal protein globally , with aquaculture production rising dramatically over the past decade .', 'Saprolegnia is a fungal-like oomycete and one of the most destructive fish pathogens , causing millions of dollars in losses to the aquaculture industry annually .', 'Saprolegnia has also been linked to a worldwide decline in wild fish and amphibian populations .', 'Here we describe the genome sequence of the first animal pathogenic oomycete and compare the genome content with the available plant pathogenic oomycetes .', 'We found that Saprolegnia lacks the large effector families that are hallmarks of plant pathogenic oomycetes , showing evolutionary adaptation to the host .', 'Moreover , Saprolegnia harbors pathogenesis-related genes that were derived by lateral gene transfer from the host and other animal pathogens .', 'The retrotransposon LINE family also appears to be acquired from animal lineages .', 'By transcriptome analysis we show a high rate of allelic variation , which reveals rapidly evolving genes and potentially adaptive evolutionary mechanisms coupled to selective pressures exerted by the animal host .', 'The genome and transcriptome data , as well as subsequent biochemical analyses , provided us with insight in the disease process of Saprolegnia at a molecular and cellular level , providing us with targets for sustainable control of Saprolegnia .']
['genome sequencing', 'genome expression analysis', 'genome databases', 'emerging infectious diseases', 'microbial pathogens', 'comparative genomics', 'biology', 'genomics', 'microbiology', 'host-pathogen interaction', 'parasitology', 'pathogenesis']