Distinctive Expansion of Potential Virulence Genes in the
Genome of the Oomycete Fish Pathogen Saprolegnia
Rays H. Y. Jiang1., Irene de Bruijn2¤a., Brian J. Haas1., Rodrigo Belmonte2,3, Lars Lo ¨bach2,
James Christie2,3, Guido van den Ackerveken4, Arnaud Bottin5, Vincent Bulone6, Sara M. Dı ´az-Moreno6,
Bernard Dumas5, Lin Fan1, Elodie Gaulin5, Francine Govers7,8, Laura J. Grenville-Briggs2,6, Neil R. Horner2,
Joshua Z. Levin1, Marco Mammella9, Harold J. G. Meijer7, Paul Morris10, Chad Nusbaum1, Stan Oome4,
Andrew J. Phillips2, David van Rooyen2, Elzbieta Rzeszutek6, Marcia Saraiva2, Chris J. Secombes3,
Michael F. Seidl8,11, Berend Snel8,11, Joost H. M. Stassen4, Sean Sykes1, Sucheta Tripathy12, Herbert van
den Berg2, Julio C. Vega-Arreguin13, Stephan Wawra2, Sarah K. Young1, Qiandong Zeng1, Javier Dieguez-
Uribeondo14, Carsten Russ1", Brett M. Tyler12¤b", Pieter van West2*"
1Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America, 2Aberdeen Oomycete Laboratory, School of Medical Sciences, University of
Aberdeen, Aberdeen, United Kingdom, 3Scottish Fish Immunology Research Centre, School of Biological Sciences, University of Aberdeen, Aberdeen, United Kingdom,
4Plant-Microbe Interactions, Department of Biology, Utrecht University, Utrecht, The Netherlands, 5Universite ´ de Toulouse; UPS; Laboratoire de Recherche en Sciences
Ve ´ge ´tales, Castanet-Tolosan, France and CNRS, Laboratoire de Recherche en Sciences Ve ´ge ´tales, Auzeville, Castanet-Tolosan, France, 6Division of Glycoscience, School of
Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, Stockholm, Sweden, 7Laboratory of Phytopathology, Wageningen University,
Wageningen, The Netherlands, 8Centre for BioSystems Genomics, Wageningen, The Netherlands, 9Dipartimento di Gestione dei Sistemi Agrari e Forestali, Universita `
degli Studi Mediterranea, Reggio Calabria, Italy, 10Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio, United States of America,
11Theoretical Biology and Bioinformatics, Department of Biology, Utrecht University, Utrecht, The Netherlands, 12Virginia Bioinformatics Institute, Virginia Polytechnic
Institute and State University, Blacksburg, Virginia, United States of America, 13ENES Unidad Leo ´n, Universidad Nacional Auto ´noma de Me ´xico, Leo ´n, Mexico,
14Departamento de Micologı ´a, Real Jardı ´n Bota ´nico CSIC, Madrid, Spain
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.
Citation: Jiang RHY, de Bruijn I, Haas BJ, Belmonte R, Lo ¨bach L, et al. (2013) Distinctive Expansion of Potential Virulence Genes in the Genome of the Oomycete
Fish Pathogen Saprolegnia parasitica. PLoS Genet 9(6): e1003272. doi:10.1371/journal.pgen.1003272
Editor: John M. McDowell, Virginia Tech, United States of America
Received August 17, 2012; Accepted December 10, 2012; Published June 13, 2013
Copyright: ? 2013 Jiang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Agriculture and Food Research Institute of the National Institute of Food and Agriculture, grant 2008-35600-04646 (to
BMT); by the Ministerio de Ciencia e Innovacio ´n, Spain (CGL2009-10032) (JD-U); and by the BBSRC, NERC, The Royal Society, the University of Aberdeen, and the
European Union (FP7) for a Marie Curie Initial Training Network award ‘‘SAPRO’’ (PvW). The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
¤a Current address: Laboratory of Phytopathology, Wageningen University, Wageningen, The Netherlands.
¤b Current address: Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon, United States of America.
. These authors contributed equally to this work.
" These authors also contributed equally to this work.
PLOS Genetics | www.plosgenetics.org1 June 2013 | Volume 9 | Issue 6 | e1003272
Saprolegnia species are watermolds or oomycetes that are
endemic to probably all fresh water ecosystems. These under-
studied 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 Informa-
tion). 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 Saprolegnia-
sis), 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 . In
addition to the damage to the aquaculture industry, the declines
of natural salmonid populations have also been attributed to
Saprolegnia infections . 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, Eu-
glenozoa and Mycota, as well as in numerous bacterial lineages.
Oomycetes such as Saprolegnia belong to the kingdom Strameno-
pila (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 Peronospor-
omycetidae. 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 (Sapro-
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 , the soybean root rot pathogen
Phytophthora sojae , and the sudden oak death pathogen
Phytophthora ramorum . Sequenced and assembled genomes
have been generated for P. sojae and P. ramorum , P. infestans
, and for several relatives including the broad host range
plant pathogens Phytophthora capsici  and Pythium ultimum ,
the downy mildew pathogen of Arabidopsis, Hyaloperonospora
arabidopsidis , and the white blister rust of Brassicaceae,
Albugo candida . 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,
[7,11,19,20]. These classes of effector proteins contain amino
acid sequence motifs (RXLR, CHXC, and LFLAK respectively)
thatare involvedin entry
[12,21,22,23,24,25] and effector domains that target diverse host
physiological processes to suppress immunity and promote
infection . 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 . In the obligate biotroph H.
arabidopsidis, most gene families are smaller, especially those
encoding hydrolytic enzymes . Interestingly, EST libraries
from the saprolegniomycete plant pathogen A. euteiches revealed
the presence of Crinkler effectors (but neither NLP toxins nor
RXLR effectors) , suggesting that Crinklers are ancestral to
oomycete pathogens .
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 , 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 demon-
strated to enter fish cells through binding to a tyrosine-O-sulfated
fish cell surface ligand .
intothe host plantcell
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 evolu-
tionary 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 transcrip-
tome 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.
The Genome Sequence of S. parasitica
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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
Figure 1. Taxonomy and ancestral genomic features in S. parasitica. (A) Animal pathogenic and plant pathogenic oomycetes reside in
different taxonomic units. (B) Comparison of intron number in phytopathogenic oomycetes (the average count from the total genes of P. infestans, P.
ramorum, P. sojae, Py. ultimum and H. arabidopsidis) and S. parasitica among all genes. (C) Significant difference in intron number in 4008 orthologous
genes shared by S. parasitica and Phytophthora species (average intron count of P. infestans, P. sojae and P. ramorum). (Wilcoxon test, p,0.001). (D)
Large number of chitinase genes belonging to CAZy family GH-18 in S. parasitica (red) compared to other oomycetes (black; Ps=P. sojae, Pr=P.
ramorum, PITG=P. infestans, Hp=H. arabidopsidis, Pyu=Py. ultimum, ALNC=A. laibachii). The phylogenetic tree was constructed with chitinase genes
from oomycetes using Maximum likelihood method.
The Genome Sequence of S. parasitica
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mechanisms for invasion and colonization of animal host cells by
A compact, highly polymorphic genome exhibiting
extensive loss of heterozygosity
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 polymor-
phisms (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 heterozy-
gosity (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  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)  to 65 Mb (P. ramorum) , far below the outlier of
240 Mb (P. infestans)  (Table 1).
Approximately one-third of the assembled S. parasitica genome
was found to correspond to regions exhibiting loss of heterozy-
gosity (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 . 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
. Of these 4215, S. parasitica shares only 3518 orthologs
Table 1. Genome statistics and intron features of oomycetes.
Estimated genome size63 Mb240 Mb95 Mb 65 Mb 99 Mb 45 Mb
Total contig length42.3 Mba
G+C content 58% 51% 54%54%47% 52%
Number of genes17,06517,79716,98814,451 14,567 15,291
Gene density kb/per gene2.6 10.74.6 126.96.36.199
Gene Length mean 1521 bp1525 bp1614 bp 1624 bp1113 bp1503 bp
Genes with introns73% 67%56%53%49% 62%
Mean exon number per gene4 2.8 2.62.6 2.02.6
Exon length mean 337 bp475 bp 536 bp 552 bp 493 bp 502 bp
Intron length mean 75 bp125 bp124 bp123 bp 150 bp 121 bp
aTotal contig length adjusted for the regions of haplotype assemblies.
bGenome statistics derived from publication of these genomes.
cMeasured by repeatMasker with de novo RepeatScout.
dThe repeat content in the assembled sequence is listed in the brackets.
The Genome Sequence of S. parasitica
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(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 .
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 .
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 . 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
Saprolegnia parasitica has a very large kinome
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 , 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
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  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 .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_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 .
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 , two of the
newly identified S. parasitica CHS gene products (SPRG_09812
and SPRG_04151) contain a so-called MIT (Microtubule Inter-
acting 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 . 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).
Adaptations of metabolism to animal pathogenesis
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.
The Genome Sequence of S. parasitica
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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 indepen-
dently in the obligate oomycete plant pathogen H. arabidopsidis
, 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).
Other metabolic differences with oomycete plant
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 . 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  which has been shown to
lead to the synthesis of fucosterol in A. euteiches .
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
Candidate virulence proteins
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/; ), 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 pro-
teins (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
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
Table 2. Gene families potentially involved in pathogenesis
in Saprolegnia parasitica.
Spa Pinf PsojPram Hpa Pult Aphb
RXLR0 563 396374 1340
Crinklers0 196 100 19 20 26
NPP1-like proteins0 2739 59107
Elicitin and elicitin like2950 66 6017 45
Pectin methyl esterases0 11 19 1330
Glycosyl hydrolase74 147 183 16253 66
Pectate lyases0 3320 246 14
Polygalacturonases2 23 25 1632
13118 152 17
406 11 1134
Protease inhibitors, all7 34 24 163 15
Proteases, all270194 185 190 143200
Serine proteases76 45 3241 3434
Metalloproteases 69 51 50 4948 51
Cysteine proteases85 67 7169 33 77
ABC transporter, all 129161 186183 55136
Kinases543 444 436432271 166
aSpa=Saprolegnia parasitica, Pinf=Phytophthora infestans, Psoj=P. sojae,
Pram=P. ramorum, Hpa=Hyaloperonospora arabidopsidis, Pult=Pythium
ultimum, Aph=Aphanomyces euteiches.
bThe presence of the protein families were searched in the Aphanomycete EST
database by BLASTP. A positive hit (E value,1e-8 sequence similarity .30%) is
indicated by +.
cLectin and lectin-like families.
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Figure 2. Metabolic adaptations to animal pathogenesis. (A) Independent degeneration of nitrite and sulfite metabolic pathways in animal
pathogens and obligate biotrophic plant pathogens. Red cross indicates the gene encoding the enzyme is absent in the genome. (B) Lineage specific
expansion of amino acid transporters. Members from Pythium (black), Hyaloperonospora (green), Albugo (blue) and S. parasitica (red) are included. -
The S. parasitica-specific clade is marked with red dots. (C) Secreted peptidase families in S. parasitica and phytopathogenic oomycetes (the average
count from the total peptidase genes of P. infestans, P. ramorum, P. sojae, Py. ultimum and H. peronospora) . Peptidase_C1, Peptidase_S8 and
Peptidase_S10 are the largest families in S. parasitica. (D) Lineage-specific expansion of peptidase_C1 family. Members from P. sojae, P. ramorum and
P. infestans (black) and S. parasitica (red) are included. The S. parasitica-specific clade is marked with red dots.
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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 . The saprolegniomycete
plant pathogen Aphanomyces euteiches also lacked CBEL proteins
Figure 3. Specialized proteins in the secretome of S. parasitica. (A) Distributions of major classes of specialized secreted proteins compared
between animal and plant pathogenic oomycetes. P. infestans represents Phytophthora species. (B) S. parasitica secreted proteins that carry various
lectin domain fusions are schematically drawn. Domains or domain architectures unique to S. parasitica are marked with an asterisk. Proteins
containing single domains are also listed. (C) Phylogenetic relationship of lectins. The S. parasitica disintegrin gene (SPRG_01285 groups with
bacterial homologs; gal_lectin gene (SPRG_05731)) groups with animal species. All other paralogous S. parasitica disintegrin and gal_lectin genes
group closely with these two representatives, respectively, and are not shown.
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Protease activities secreted by Saprolegnia parasitica
We investigated whether culture filtrates containing secreted
proteins from S. parasitica could degrade trout immunoglobulin M
(IgM) as previously found for bacterial fish pathogens .
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
Figure 4. Rainbow trout IgM proteolysis by S. parasitica secreted proteases. (A) 7-day old culture filtrates were capable of degrading
rainbow trout IgM after an overnight incubation at 10uC. (B) Schematic drawing of the domains present in the protease SPRG_14567 (C) Expression
pattern of SPRG_14567 in different life stages. The RKPM of RNA-seq data is plotted, and the previously identified effector SpHTP-1 is plotted to show
contrasting expression patterns. (D) The recombinant subtilisin-like protease SPRG_14567 was partially purified through tandem ion exchange (SO32)
and nickel affinity columns (Fractions 1 to 4) following detection in a Western blot using anti-(His)5HRP antibody. (E) Fractions 2, 3 and soluble
proteins from untransformed E. coli were tested for IgM-degrading activity with only the fraction containing the recombinant SPRG_14567 exhibiting
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degrading fish IgM and could be a virulence factor that combats
the activity of fish immunoglobulins against Saprolegnia.
Candidate effector proteins
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  and entry required the N-terminal leader sequence
. 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. Proteina-
ceous PAMPs found in plant pathogenic oomycetes, namely
CBM1 , elicitins, and Cys-rich-family-3 proteins  are
found in S. parasitica (Figure S7A). Elicitins are extracellular lipid
transfer proteins that elicit defense responses in some species of
plants, especially Nicotiana . 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 
(Figure S7B). We also detected six YxSL[RK] containing
secreted proteins, previously identified as candidate effectors
, 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 .
Elevated nucleotide substitution rate of pathogenesis
associated gene families
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 substitu-
tions 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 . 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 haemo-
lysins, 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). Interest-
ingly, 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 signifi-
cant elevations in Ka/Ks as compared to the core ortholog group
Potential role of horizontal gene transfer in development
Horizontal gene transfer events from bacteria and archaea
appear to have contributed to some of the novel biosynthetic
pathways found in the oomycetes . By utilizing codon
usage and protein domain classification (see Methods), at least
100 genes could be identified with a potential phylogenetic
origin outside the super-kingdom of Chromalveolates by using
the program Alien_hunter  and by interrogating Pfam
search results. Among these, we further identified around 40
genes belonging to 5 families that could potentially be
associated with pathogenesis (Table 3). Many of these genes
appear to have been acquired from bacterial species, in
particular from Proteobacteria. Some potential acquisitions
may have occurred relatively recently as they only occur in
Saprolegnia and seem to have nucleotide compositions predictive
of foreign acquisition (Alien_hunter score .50). A caveat for
our analysis is that S. parasitica is the only available genome so
far outside of the plant pathogenic oomycetes. There are a
great variety of basal oomycetes [56,57] that do not have
genome sequence information and have not been investigated.
Therefore, it could be that these HGT events could have
occurred in some ancestral oomycetes .
Several groups of extracellular enzymes were potentially
acquired from bacteria; for example, the CHAP (cysteine,
histidine-dependent amidohydrolases/peptidases) family and a
family of secreted nucleases (Table 3). The presence of the
CHAP family (pfam hit E value,1e-5) is unexpected in S.
parasitica, because it is commonly associated with bacterial
physiology and metabolism . These genes have undergone
repeated duplications in S. parasitica, resulting in an expansion
of gene numbers. Another potential bacterial acquisition is a
family of toxin-like proteins similar to haemolysin (HlyE), a
pore-forming toxin from enterobacteria such as Salmonella .
These genes have also undergone recent duplication resulting
in nine copies in S. parasitica. Two members (SPRG_03140,
SPRG_20514) of the HlyE family are expressed during
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Another distinctive feature of the S. parasitica secretome is the
presence of animal-like surface proteins. The phylogenetic
affinities of the two groups, distinguished by gal_lectin-like and
disintegrin-like domains, suggest a possible origin via HGT, but
from different sources. Gal_lectin refers to the D-galactoside
binding lectin initially purified from the eggs of sea urchin
[59,60,61], representing a group of lectins that occurs widely on
fish eggs and skins . Phylogenetic analysis shows that the
closest homologs are animal gal_lectins (Figure 3C). The S.
parasitica gal_lectin genes are among the most highly induced and
highly expressed genes in pre-infection and infection stages,
suggesting that gal_lectin may facilitate adhesion and invasion of
fish cells. The gal_lectin genes contain an unusually large number
of introns (7 introns), as do mammalian and fish gal_lectin genes,
further suggesting a common origin. The S. parasitica genes have a
codon usage similar to core orthologs, in contrast to other
candidate HGT genes, suggesting they have been in the S.
parasitica genome long enough to be largely assimilated. The S.
parasitica disintegrin genes were potentially acquired from bacteria
(Figure 3C, Table 3) and have since expanded. Disintegrins were
initially identified as proteins preventing blood clotting in viper
venoms . In animals, disintegrins inhibit aggregation of the
platelets by binding to the integrin/glycoprotein IIb-IIIa receptor.
The crucial amino acid motif CRxxxxxCDxxExC, mediating
ligand binding, is conserved in the S. parasitica disintegrins. All 16
disintegrin genes in S. parasitica are expressed in the pre-infection
stages (see below), with several members belonging to the top 1%
most highly expressed genes, suggesting that they may play a role
interacting with animal hosts. However, we have not been able to
experimentally demonstrate the role of disintegrins in pathogenesis
so far (Text S1 and Figure S10).
Tissue-specific and host-induced gene expression
S. parasitica has, like most other oomycetes, clearly defined life
stages, including motile zoospores that are able to swim, encyst
and germinate upon attachment to its host tissue. We performed
strand-specific Illumina RNA-Seq analysis of 4 developmental
stages (mycelium, sporulating mycelium, cysts, and germinating
cysts [3–5 hours]) of S. parasitica, as well as a time course analysis
(0, 8 and 24 hours) of a rainbow trout fibroblast cell-line (RTG-2)
challenged with cysts of S. parasitica. RNA-Seq reads were mapped
to the S. parasitica genome and gene annotations, and transcript
abundance values were quantified as RPKM (reads per kilobase
transcript length per million reads mapped).
Large numbers of genes were differentially expressed in the
various life stages and conditions. Cyst and germinating-cyst stages
showed similar expression profiles (linear correlation R2=0.93,
p,0.001), with less than 6% of all the genes showing .4-fold
expression differences (p,0.001; Figure 5A). In contrast, the other
developmental life stages and infection time course showed
somewhat larger differences (R2of 0.84–0.88), with up to 27%
of genes differentially expressed between cysts and mycelia (.=4-
fold difference; p,=0.001). The time course experiment shows
the relative abundance of the host and pathogen transcripts
changing as infection progressed (Figure 5B). At the 0 hour and
8 hours time points, very few pathogen transcripts were detected
(less than 1% and 3%, respectively) whereas at 24 hours, 63% of
the transcripts were derived from the pathogen. At 24 hours, the
transcript profile of the pathogen closely resembled that of in vitro-
grown mycelia, with only 3.5% of all genes differentially induced
between the two stages (.4 fold differences, p,0.001). Compared
to the cysts used as inoculum, 7.2% of genes were induced after
8 hours of infection and 10% of genes were induced by 24 hours
(.4 fold differences, p,0.001) (Figure S11A). We have defined the
stage prior to host tissue colonization (germinating cyst) as the pre-
infection stage. In plant pathogenic oomycetes, germinating cysts
express many pathogenesis-associated genes [12,64,65,66,67]. The
previously characterized S. parasitica gene encoding the host
targeting protein SpHtp1  is induced more than 100-fold in
the pre-infection stage as compared to other stages. At the pre-
infection stage, 10% of all genes were induced as compared to the
mycelial stage (.4 fold differences, p,0.001) (Figure 5C; Figure
S11B; Table S12). The profile of the germinating cysts - resembled
that of the 8 hours fish cell infection (R2=0.88, p,0.001), with
only 4% of genes showing induction (.4-fold differences,
For genes that encode proteins specific to or expanded in S.
parasitica as compared to other oomycetes, around 25% were
induced in germinating cysts compared to mycelia (.4-fold
differences, p,0.001) (Figure S11C, Table S13). A total of 14
protein families showed both lineage-specific domain expansion
and up-regulation in germinating cysts (Figure 5D). The largest
groups are proteins carrying ankyrin domains or lectin domains,
which suggests the importance of protein-protein and protein-
carbohydrate interactions in the initial stages of host colonization.
Another group of proteins belonging to this category are
transporters such as ion transporters and sodium symporters,
suggesting that metabolic exchange processes are active during the
establishment of early infection.
The RNA-Seq data also suggest that the very large S. parasitica
kinome plays an important role during the infection process.
Around 10% of all kinase genes, spanning all major kinase
Table 3. Predicted horizontally transferred genes that may be associated with pathogenesis in Saprolegnia parasitica.
Pfam function Functional Description
in the family
Disintegrin Disintegrinproteobacteria16 SPRG_14051 secretedrecent
Laminin like Associated with cell surface-1 SPRG_08424 secretedrecent
CHAP CHAP domain-7 SPRG_15528secreted
Endonuclease DNA/RNA non-specific
HylE Haemolysin E enterobacteria9 SPRG_04818membrane recent
aSubcellular localization is predicted by the N-terminal signal peptide, mitochondrial targeting motif and transmembrane domains.
bThe time of horizontal gene transfer is estimated by the presence in other oomycetes and coding potential of a given gene. ‘a recently acquired gene’ refers to a gene
occurring only in Saprolegnia and having an uncharacteristic coding potential.
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classification groups, showed 4-fold or more induction in
germinating cysts compared to mycelia (.4-fold differences,
p,0.001) (Figure S11C). Many of the numerous S. parasitica
proteases were expressed in a specific life stage or distinct point
during the infection process (Figure 5D/E, Table S14). One
group of peptidases (6%) was expressed in germinating cysts.
These include subtilase proteins (SPRG_15005) that carry ricin
lectin domains and are highly expressed in germinating cysts. A
large group of peptidases (19%) were induced during the
interaction with fish cells. There are 7 protease-inhibitors
encoded in the genome; and they also showed patterns of
differential expression (Figure 5C). The Kazal peptidase inhibitor
(SPRG_09563) was most highly expressed in cysts and germi-
Figure 5. Differentially expressed genes detected by RNA-Seq. (A) Percentage of differentially expressed genes in pair-wise comparisons of
tissue types. Genes with 4 fold RPKM (reads per kilobase per million) differences were considered to be differentially expressed (negative binomial
exact test p,0.001, p value adjusted with Benjamini & Hochberg correction, Table S12) (B) Gene families showing differential expression between
vegetative tissue (mycelia and sporulating mycelia) and pre-infection tissues (cysts and germinating cysts). CBEL:fungal Cellulose Binding Domain Like
protein, EGF:(Epidermal Growth Factor, gal_lectin: Galactose binding Lectin domain, HST: Heat shock factors, PLAC8: Placenta-specific gene 8 protein,
SDF: Sodium Dicarboxylate symporter Family (Table S13). (C) Growth phase specific expression of peptidases and protease inhibitors (Table S14). (D)
Relative abundance of S. parasitica and fish transcripts during interaction. (E). S. parasitica transcript distribution in pre-infection versus vegetative
tissue. logFC=log2(pre-infection/vegetative/pre-infection); logConc=the log2 of average reads counts per million of each gene in the two tissue
types,). Red dots indicate significant differences (p,0.001; negative binomial test).
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To confirm the RNA-seq data, we have also performed qPCR
for a set of disintegrin genes, showing expression in cysts and
germinating cysts, but no detection in mycelium and sporulating
mycelium as seen for the RNA-seq data (Figures S11 C,D).
Taken together, the RNA-seq data reveal that members of the
kinases, proteases, disintegrins and gal_lectins show upregulated
expression in the pre-infection stage (Table S13 and S14), which
warrants using these candidate genes for future pathogenesis
The genome sequence of S. parasitica, together with transcrip-
tome and polymorphism data, reveals a predicted core proteome
very similar to those of plant pathogenic oomycetes, but an
adapted proteome strongly aligned to its animal-pathogenic
From the genome sequence and the RNA-Seq data we could
identify several groups of proteins predicted to be secreted at the
pre-infection stages that might facilitate early interactions with the
hosts of S. parasitica. Several of these proteins seem to be unique to
S. parasitica and may have evolved to interact specifically with fish
cells. They are predicted to be targeted to the extracellular
environment or incorporated into the exterior surface of the
pathogen. Several of the proteins contain CBM domains (fungal
cellulose binding domain), ricin-like domains, Notch-like domains
and/or various peptidase domains. Most putative early interaction
proteins have potential roles in pathogenesis, such as animal-cell-
surface-like proteins (disintegrin, gal_lectin) and haemolysin E
toxin-like proteins. Lectins could help cysts to bind to host skin.
Since the lectins are highly expressed during the initial and later
infection stages, we hypothesize that they play an important role in
cell-cell contact throughout the interaction. This would also
suggest that an intimate contact with the host cell is required for
pathogenesis, suppression of host defence processes, and/or
nutrient uptake. Following attachment, the pathogen engages a
large arsenal of potential virulence factors in the form of proteases
to attack the host tissue. Interestingly, S. parasitica has one of the
largest numbers of proteases found in any organism. At least one
protease (SPRG_14567) was found to be able to degrade IgM,
suggesting an active role in suppressing initial immune responses,
as fish IgM’s are able to bind to infection related antigens, even in
the absence of prior immunization.
In comparison to plant pathogenic oomycetes, S. parasitica has
no canonical RXLR or Crinkler effector genes, nor NLP toxin
genes in its genome. Nevertheless, one small protein family has
been found for which one member was shown to translocate inside
trout cells [2,30], which suggests that the interaction of S. parasitica
with its hosts is more subtle than a simple necrotrophic interaction
based on secretion of toxins and protein degradation. In fact, we
speculate that the initial stages of the interaction may involve a
more ‘biotrophic’ approach by the pathogen, whereby the
immune response of the host is avoided or even suppressed during
initial colonization via, for example, proteases or effector proteins.
Following this biotrophic stage, the host tissue is bombarded with
proteases, lipases, and lysing enzymes. If S. parasitica was a plant
pathogen, it would thus have been classified as a ‘hemi-biotroph’
and not a saprotroph as its name would suggest. Further
experiments are required to demonstrate that this is indeed the
Pathogenicity towards animals has evolved independently in
both the fungi and oomycetes. It has also evolved at least three
additional times within the kingdom Stramenopila: within the
Pythiales (Figure 1A), within the genus Aphanomyces  and in the
non-oomycete Blastocystis. Analyses of the genomes or sequenced
ESTs of these other pathogens reveals some interesting parallels.
For example, based on only a small set of ESTs of the oomycete
human pathogen Py. insidiosum , an identified expressed lectin
CBM-encoded transcript has been implicated in the process of
pathogenesis. Blastocystis hominis is a Stramenopile human pathogen
with a very small genome of 19 Mb encoding only 6020 genes,
and is phylogenetically very divergent from oomycetes . Despite
the large evolutionary distance, the genome of Blastocystis shows
reduction of nitrogen and sulfite metabolic pathways, similar to
what is seen in obligate plant pathogens. In addition, similar to the
acquired lectins in S. parasitica, Blastocystis has also horizontally
transferred genes with animal like features, such as genes encoding
Ig domains . The fungal pathogen Batrachochytrium dendrobatidis
that causes global amphibian decline, inhabits an aquatic
environment like S. parasitica, and also causes diseases on animal
skin and tissues. Analysis of its genome revealed patterns of
expansion of protease families . Although the particular
families are different, B. dendrobatidis possesses several families of
lineage expanded proteases such as metallopeptidase (M36).
The extensive LOH observed in S. parasitica, covering approx-
imately one-third of the genome and also the high rate of
polymorphism (2.6%) in the remainder of the genome are similar
to recent observations in the genome of the oomycete plant
pathogen Phytophthora capsici, where LOH was found to be
associated with changes in mating type and pathogenicity .
We speculate that, as in P. capsici, LOH may provide a mechanism
in S. parasitica for rapidly expressing diversity within a population,
fixing alleles, and enabling rapid adaptation to its environment.
The evolution of plant and animal pathogenesis in oomycetes
has been associated with several major molecular events
(Figure 6). Since both Phytophthora and Saprolegnia have large
kinomes, the expansion of kinases is likely a relatively early event
in oomycete evolution. The comparison of the animal pathogen
S. parasitica with plant pathogens with different lifestyles has
shown that surviving on ammonium rich tissue has led to a
reduction of metabolic pathways independently in S. parasitica and
obligate oomycete plant pathogens. Similar reductions have also
occurred in obligate fungal pathogens  and in the distantly
related stramenopile human pathogen Blastocystis . The
evolution of plant pathogenicity has been associated with a series
of reduction events such as intron loss, chitin loss and sterol loss.
Some of these losses may be due to evasion of plant immunity; for
example chitin can act as a PAMP to trigger plant defense
responses. The evolution of plant pathogens has been accompa-
nied by expansions of large repertoires of effectors, which have
been shown to modulate plant host physiology. In contrast, the
development of animal pathogenesis has been facilitated by
expansion of proteases and horizontally acquired lectins and
Strain selection and genomic DNA isolation
Saprolegnia parasitica isolate CBS223.65 was isolated from young
pike (Esox lucius) in 1965 and obtained from Centraal Bureau voor
Schimmelcultures (CBS), the Netherlands. Saprolegnia parasitica
isolate VI-02736 (N12) was obtained from parr of Atlantic salmon
in Scotland in 2002 (Lochailort)  and kindly provided by Dr.
Ida Skaar (Norwegian Veterinary Institute). Both isolates were
maintained on potato dextrose agar (Fluka). For genomic DNA
isolation, S. parasitica was grown for three days at 24uC in pea
broth (125 g L21frozen peas, autoclaved, filtered through cheese
cloth, volume adjusted to 1 L and autoclaved again). Genomic
The Genome Sequence of S. parasitica
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DNA extraction was adapted from Haas et al . Fresh mycelia
(,1 g) were ground to a fine powder under liquid nitrogen,
mixed with 10 mL of extraction buffer (0.2 M TrisHCl pH 8.5,
0.25 M NaCl, 25 mM EDTA, 0.5% SDS), 7 mL Tris-equili-
brated phenol and 3 mL of chloroform:isoamyl alcohol (24:1),
incubated at room temperature for 1 hr and centrifuged at
6,000 g for 30 m. The aqueous phase was extracted with equal
volume of chloroform:isoamyl alcohol (24:1) and centrifuged at
10,000 g for 15 min. 50 ml of 10 mg/ml RNase A was added to
the aqueous phase and incubated for 30 min at 37uC. Isopro-
panol (0.6 volumes) was added, mixed gently, and then the DNA
was precipitated on ice for 30 min. DNA was collected by
centrifugation at 10,000 g for 20 min, washed with 70% ethanol,
dried and resuspended in DNase/RNase-free water. DNA was
checked for quality and RNA contamination by gel electropho-
resis using 0.8% agarose.
Collection of samples and RNA isolation
The life stages of S. parasitica were harvested as described by van
West et al. . Zoospores and cysts were collected by pouring the
culture filtrate through a 40–70 mm cell strainer and concentrated
by centrifugation for 5 min at 1500 g. RNA was isolated from
mycelia and sporulating mycelia using the RNeasy kit (Invitrogen)
according to the manufacturer’s protocol. RNA from zoospores,
cysts and germinating cysts was resuspended in TRIzol (Invitro-
gen) and aliquoted as 1 ml portions in 2-ml screw-cap tubes
containing 10–35 glass 1 mm diameter beads (BioSpec) and
immediately frozen in liquid nitrogen. Frozen cells were processed
with a FastPrep machine (ThermoSavant) and shaken several
times at a speed of 5.0 for 45 sec until defrosted and homogenized.
TRIzol RNA isolation was then performed according to manu-
facturer’s recommendations (Invitrogen).
Host-induced gene expression was measured in rainbow trout
(Oncorhynchus mykiss) gonadal tissue continuous cell line RTG-2,
obtained from the American Type Culture Collection (ATCC
CCL-55) . RTG-2 cells were grown to a monolayer at 24uC
(no CO2) and washed twice with Hank’s Balanced Salt Solution
(HBSS). Cells were harvested in approximately 5 ml of HBSS
and S. parasitica cysts were added (56104cysts per 75 cm flask)
and the mixture was then incubated at room temperature for
15 min. At this time, the time 0 hr (T0) stage sample was
collected, by gently pouring off medium (and also loose/non-
attached cysts) and then adding TRIzol reagent for RNA
extraction. Other samples were incubated for 8 hr (T8) or
24 hr (T24) at 24uC (no CO2) in Leibovitz (L-15) growth medium
(Gibco) supplemented with 10% foetal calf serum (Biosera),
200 U ml21penicillin and 200 mg ml21streptomycin (Fisher).
Cells were harvested for RNA extraction by pouring off medium
and adding TRIzol. Cells were loosened using a cell scraper
(Fisher) and the suspension was aliquoted as 1 ml portions into 2-
ml screw-cap tubes containing 10–35 glass 1 mm diameter beads
(Biospec) and immediately frozen in liquid nitrogen. Frozen cells
were processed using a FastPrep machine and shaken several
times at a speed of 5.0 for 45 sec until defrosted and
Figure 6. Molecular genetic events associated with the evolution of animal and plant pathogenesis in oomycetes. The lineages of
animal pathogens are colored red and the lineages of plant pathogens are colored green. The basal lineage is colored brown. S and N pathways refer
to sulfite and nitrite assimilation, respectively.
The Genome Sequence of S. parasitica
PLOS Genetics | www.plosgenetics.org14 June 2013 | Volume 9 | Issue 6 | e1003272
homogenized. TRIzol RNA isolation was then performed
according to manufacturer’s recommendations.
Infected and uninfected fish tissue was collected from a trout (O.
mykiss, caught in a commercial Scottish hatchery) that showed
lesions caused by S. parasitica infection. Tissue was collected from
the lesion (body) and from an infected anal fin. As an uninfected
control, similar tissue not showing symptoms of infection was
collected from the same fish. Tissue was ground up in liquid N2
and RNA was isolated using the RNeasy kit (QIAGEN) according
to manufacturer’s recommendations.
Infected and uninfected Atlantic salmon (Salmo salar) egg
samples were collected from a commercial Scottish hatchery. Five
eggs per sample were ruptured using a needle and placed into 5 ml
of TRIzol and RNA was isolated according to manufacturer’s
recommendations with an additional phenol-chloroform extrac-
tion step. Multiple sample batches were pooled to obtain sufficient
material for RNA-Seq library construction.
All RNA samples were resuspended in DNase/RNase-free
water and treated with Turbo DNA-free DNase (Ambion)
according to manufacturer’s recommendations. RNA was checked
for quantity and purity using a Nanodrop spectrophotometer
(Thermo Scientific) and 1% agarose gel electrophoresis. Samples
were stored at 280uC and were not defrosted until use.
454 and Sanger sequencing for genome assembly
454 fragment and 3 kb jumping whole genome shotgun
libraries were generated for isolate CBS 223.65 as previously
described . Libraries were sequenced with ,400 base single
end reads (Titanium chemistry) using a 454 GS FLX sequencer
following the manufacturer’s recommendations (454 Life Sci-
ences/Roche). Approximately a total of 22-fold sequence
coverage was generated from fragment and 3 kb jumping
libraries combined. A 40 kb insert Fosmid whole genome
shotgun library from isolate CBS 223.65 was generated using
the EpiFOS fosmid cloning system following manufacturer’s
recommendations (Epicentre). The Fosmid library was end-
sequenced with ,700 bp reads using Sanger technology to
approximately 0.3-fold coverage using a 3730xl DNA analyzer
following manufacturer’s recommendations (Applied Biosys-
Illumina sequencing for variant detection and RNA-Seq
For variant calling, Illumina whole genome shotgun fragment
libraries were generated for isolates CBS 223.65 and VI-02736 as
previously described  and sequenced with 76 base paired-end
reads to a minimum of 70-fold sequence coverage using an
Illumina Genome Analyzer II (Illumina) following the manufac-
turer’s recommendations. Illumina strand-specific dUTP RNA-
Seq libraries were generated for all RNA samples as previously
described  with the following modifications. The mRNA was
processed using the Dynabeads mRNA purification kit (Invitrogen)
and incubated with RNA fragmentation buffer (Affymetrix) at
80uC for 1.5 to 3.5 minutes depending sample quality. Indexed
adaptors for Illumina sequencing were ligated onto end-repaired,
A-tailed cDNA fragments by incubation with 4,000 units of T4
DNA ligase (New England Biolabs) in a 20 ml reaction overnight
at 16uC. 16 to 21 cycles of PCR were used to amplify sequencing
libraries. Libraries were purified using 1–3 rounds of AMPure
beads (Beckman Coulter Genomics) following manufacturer’s
recommendations. Libraries were sequenced with 76 base paired-
end reads using an Illumina Genome Analyzer II following
manufacturer’s recommendations (Illumina) generating a total of
124 million reads.
Saprolegnia parasitica CBS 223.65 de novo genome
Sanger Fosmid paired-end reads were quality trimmed using the
ARACHNE ‘Assemblez’ module (http://www.broadinstitute.org/
crd/wiki/index.php/Assemblez). Read headers were amended to
contain template and pairing information. Sanger, 454 fragment
and 3 kb jumping reads were assembled using 454’s Newbler
assembler version 04292009 (454 Life Sciences/Roche). The
resulting assembly was further processed using the ARACHNE
‘HybridAssemble’ module (http://www.broadinstitute.org/crd/
wiki/index.php/HybridAssemble) using the 454 assembly, Sanger
and 454 read data as input with option ‘RecycleBadContigs’
turned off allowing for extra copies of repeat sequences to be
assembled. The Arachne ‘AddReadsAsContigs’ module was run to
allow assembly of additional repetitive sequence. The assembly
was screened for known sequencing vector sequences using
BLAST and contigs with hits to known sequence vectors were
removed. The assembly was further post-processed by removing
contigs and scaffolds less than 200 bp and 2 kb in length
Illumina shotgun sequence data were used to identify
polymorphisms in two S. parasitica strains (CBS 223.65 and VI-
02736). Illumina paired-end fragment reads from strains CBS
223.65 and VI-02736 were independently aligned to the S.
parasitica CBS 223.65 reference assembly using the BWA aligner
 using default settings. Read alignments were sorted by
scaffold and position along the reference assembly. SNP calling
was performed using the GATK Unified Genotyper module .
Variant Call Format (VCF) files containing SNP calls were filters
for low quality using parameters: AB.0.75 && DP.40 I
DP.500 I MQ0.40 I SB.20.10. SNP calls for CBS 223.65
and VI-02736 can be retrieved from the Broad Institute
Saprolegnia parasitica genome database website (http://www.
Loss of heterozygosity and haplotype region
SNP calls and depth of read coverage information were parsed
from the VCF file described above and analyzed in non-
overlapping 5 kb windows (Figure S1). Using this information
genome segments were partitioned into three groups: separated
haplotypes (coverage depth ranging 20–55-fold and SNP rate
,1%), diploid homozygous involving LOH (coverage depth
ranging 56–90-fold and SNP rate ,1%), and diploid heterozy-
gous (coverage depth ranging 40–90-fold and SNP rate .=1%).
Coverage of separated haplotype regions peaks at ,40-fold and
the regions are mostly devoid of SNPs. The region corresponding
to the diploid consensus exhibits ,60-fold coverage and nearly a
3% SNP rate. The peaks in Figure S1B corresponding to the
separated haplotype and consensus diploid regions are connected
by a small ridge, which correspond to windows spanning
boundaries between the different kinds of regions. The coverage
for the diploid consensus regions is not exactly double as
compared to the predicted separately assembled haplotype
regions, and is less than the diploid homozygous regions; most
likely this results from the relative difficulty of aligning short
Illumina reads to diploid consensus sequences in the context of
the high polymorphism rate observed.
Individual genes located in haplotype contigs were assigned as
likely allelic pairs based on SNP rate, depth of coverage, and
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taking into consideration best reciprocal blast matches and synteny
between separately assembled haplotype contigs.
Gene finding used both evidence-based (including EST, RNA-
Seq and homology data) and ab initio methods. Gene-finding
algorithms FGenesH, GeneID and GeneMark were trained for S.
parasitica using existing gene and EST datasets. Then a statistical
sampling of gene calls as well as genes of interest were manually
curated, and the results were used to validate gene calls and fine-
tune the gene caller. RNA-Seq data was incorporated into gene
structure annotations using PASA  as described in Rhind et al.
. Subsequently, the annotated total gene set was subjected to
Pfam domain analysis, OrthoMCL clustering analysis and KEGG
metabolic pathway analysis.
Expression analysis with RNA-Seq
Illumina RNA-Seq data was processed as follows. Sequencing
adaptors were identified and removed from reads by exact match
to adaptor sequences. Reads were aligned to S. parasitica gene
transcripts using Bowtie (allowing up to 2 mismatches per read,
and up to 20 alignments per read). Transcript levels were
calculated as FPKM (fragments per kilobase cDNA per million
fragments mapped). The program EdgeR  was used to identify
differentially expressed transcripts. Transcripts with significantly
different levels (p,=0.001 and over 4-fold difference) were
identified, and p-values were adjusted for multiple testing by using
the Benjamini & Hochberg  correction.
The predicted proteomes of S. parasitica and representative plant
pathogenic oomycetes were annotated by mapping against a
reference set of metabolic pathways from KEGG (Kyoto
Encyclopedia of Genes and genomes) . The method used,
KAAS (KEGG Automated Annotation Server), utilizes bidirec-
tional best hits to assign pathways. Subsequently, the metabolic
genes and pathways were manually annotated and compared to
other oomycete pathogens. Known genes involved in nitrogen and
sulfur metabolism were used to search the S. parasitica genome by
TBLASTN search; genes were considered to be candidates if a
positive hit was found (E value,1e-5). For chitin metabolism
analysis, genes were annotated based on Pfam homology (E
value,1e-5) with the exception of chitin synthase genes, which
were identified after Blastp analysis against a set of oomycete and
fungal CHS. A total of twelve GH18 genes were identified, of
which six are arranged in small clusters of two paralogous genes.
Horizontal gene transfer analysis
Two different approaches were used to screen S. parasitica genes
for candidate HGT origins. In the first approach, the genome
sequence was screened with the program Alien_hunter . The
program utilizes an interpolated variable order motif method to
determine horizontally transferred events, purely based on
compositional difference between a region and the whole genome
framework. Because the methodology is independent of any
existing datasets, we used it to examine the S. parasitica genome.
Genomic regions were identified as alien when the Alien_hunter
score was above 50. Out of 1442 S. parasitica supercontigs, 206
supercontigs had distinct regions marked as alien after running
Alien_hunter. Subsequently, the 1616 gene models that lay within
the candidate alien regions were extracted and compared with
other oomycete genomes (P. sojae, P. ramorum, P. infestans, H.
arabidopsidis and Py. ultimum). In the second approach, the entire
proteome of each oomycete was scanned for homology to Pfam-A
protein families using the hmmscan algorithm from Hmmer 3.0
applied to the Hidden Markov Model dataset (Pfam-A.hmm v.24).
A cut-off e-value threshold of 1e-3 was applied. From the Pfam
domain analysis we obtained 307 sequences that had distinct
domains not found in any of the Phytophthora species, and 31 of the
candidates derived from the Pfam analysis overlapped with the
results from Alien_hunter. We then blast-searched all 1616 genes
from Alien_hunter and the 307 genes from the domain analysis
against the NCBI non-redundant database (nr) to obtain the
primary functions. Phylogenetic analysis using neighbor joining
was then performed on the final set of genes.
Analysis of secreted proteases
S. parasitica CBS 223.65 mycelium plugs were grown in pea
broth for 2 or 7 days. The culture supernatants were harvested,
centrifuged at 50006g for 10 min (4uC) and the soluble fraction
used as a source of secreted proteases. The ammonium sulfate
precipitated fraction from rainbow trout serum  was used as
source of fish IgM (10 mg/mL of total protein concentration). For
protease activity experiments, 50 mL of the culture supernatants
were incubated overnight at 10uC with 5 mL of the IgM enriched
fraction. Pea broth was used as a negative control. Heat-
inactivated supernatant was obtained by incubating the culture
supernatants for 15 min at 95uC. The protease inhibitors EDTA
(10 mM), PMSF (1 mM) and E-64 (10 mM) were used to identify
classes of proteases involved in IgM degradation.
Two samples volumes (2.5 and 5 mL) were spotted onto
nitrocellulose membranes, allowed to dry for 45 min, blocked
with milk-PBS (5% dry-milk) and remaining IgM was detected
using a monoclonal anti-trout/salmon HRP conjugated antibody
(Aquatic Diagnostics). SPRG_14567 was cloned from S. parasitica
CBS223.65 cDNA into the pET21B vector. E. coli Rossetta-gami
B competent cells were transformed with the expression vector by
heat-shock. Transformed cells were grown in modified LB media
(100 mM NaHPO4, pH 7.4; 2 mM MgSO4, glucose 0.05% w/v;
and NaCl 0.5% w/v). When E. coli cells reached an OD600of 0.8,
cultures were induced with IPTG (10 mM) and incubated at
200 rpm at 37uC for 12 hr. The soluble fraction was purified from
French press supernatant by tandem ion exchange (on SO32) and
nickel affinity columns. Chromatography fractions were submitted
tor 1D SDS-gel electrophoresis (NuPAGE Electrophoresis System,
Invitrogen) and then transferred to nitrocellulose membranes
(XCell II Blot module, Invitrogen). Membranes were blocked with
milk-PBS-T (0.02% v/v Tween, 10% dry milk) for 20 min. Anti-
(His)5HRP conjugate (QIAGEN) was added to a final dilution of
1:20,000 and incubated at room temp for 1 hr for detection of the
recombinant protein. Chromatography fractions were tested for
IgM degrading activity as previously described . Briefly, 50 mL
(5 mg/mL of total protein concentration) of the fractions were
incubated overnight at 10uC with 5 mL of the IgM enriched
fraction. Untransformed E. coli soluble proteins were used as a
control. Remaining IgM was then detected in a dot-blot as
Phylogeny reconstruction and domain combination
Gene models were analyzed using TMHMM and ClustalX. For
phylogeny reconstruction, sequences were aligned using ClustalX
and aligned sequences were subjected to phylogenetic analysis (NJ)
using PAUP. The predicted proteome of S. parasitica was compared
to that of six pathogenic oomycetes together with sixty-four other
eukaryotic species covering all major groups of the eukaryotic tree
of life, as previously used by Seidl et al. . We used hmmer-3
The Genome Sequence of S. parasitica
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[http://hmmer.org] and a local Pfam-A database to predict
1,798,601 domains in 862,909 proteins of which 19,896 domains
in 10,887 proteins are found in S. parasitica. The architectures of
multi-domain proteins were analyzed from the N- to the C-
terminus, which identified 18,512 domain combinations consist-
ing of two contiguous domains. Of these there were 1120
proteins. The great majority of combinations were specific to a
single species, and only 58 combinations were found in more than
one oomycete species. S. parasitica contained 338 domain
combinations that are specific for oomycetes including 169
domain combinations encoded by 215 genes that are specific for
this species (Table S9).
Saprolegnia parasitica CBS 223.65: Sanger sequence data were
submitted to the NCBI Trace Archive (http://www.ncbi.nlm.nih.
gov/Traces/trace.cgi) and can be retrieved using query: CEN-
TER_NAME=‘‘BI’’ and CENTER_PROJECT=‘‘G1848’’. 454
and Illumina sequence data were submitted to the NCBI Short
Read Archive (SRA) (http://www.ncbi.nlm.nih.gov/sra) and can
be retrieved using the following accession numbers: 454 fragment
reads (SRX007896, SRX007901, SRX007898, SRX007895,
SRX005344, SRX007971, SRX007958, SRX005346); 454 3 kb
(SRX022535); Illumina RNA-Seq reads (BioProject 164643:
mycelium SRX155934, sporulation mycelium SRX155933, cysts
SRX155932, germinating cysts SRX155938, infected fish cell-line
t=0 SRX155937, infected fish cell-line t=8 SRX155936 and
infected fish cell-line t=24 SRX155935. BioProject 167986:
infectedfish tissue SRX155944,
SRX155942, infected salmon eggs SRX155943, SRX155940
and uninfected salmon eggs SRX155941). Draft genome assembly
sequence was submitted to GenBank (BioProject ID 36583,
Saprolegnia parasitica VI-02736 : Illumina sequence data were
submitted to the SRA (BioProject 164645, accession SRX155939).
SNP calls for strains CBS 223.65 and VI-02736 can be
downloaded from the Broad Institute Saprolegnia parasitica genome
coverage was examined at 100 base intervals, yielding peak
coverage of approximately 506 (B) Frequency of nucleotide
positions with given k-mer coverage. The mean k-mer coverage
(38.4) of haplotype alleles was calculated from the first Gaussian
curve (colored red). The mean k-mer coverage (59.7) of the single
copy sequences was calculated from the second Gaussian curve
(colored orange) fitted to the main peak. The single copy
sequences’ coverage was used to calculate the total genome size.
Relative abundance from 0 to 1 of nucleotides was plotted. The
fitted Gaussian curves have R2.0.999 (P,1e-10). (C) Illumina
read coverage and polymorphism rates averaged across non-
overlapping 5 kb genomic regions. Color bar indicates numbers of
5 kb regions. The plot shows partitioning of the genome segments
into three groups: separated haplotypes, diploid homozygous
involving LOH, and diploid heterozygous (as defined in Text S1).
(A) Illumina read coverage of the assembly. The
Phytophthora. (A) Number of genes orthologous between S. parasitica
Gene content differences between S. parasitica and
and P. infestans. The core proteome that is conserved among
multiple Phytophthora species is indicated with a dark green circle.
The phytopathogen core proteome derived from P. infestans, P.
ramorum, P. sojae, Pythium ultimum, and Hyaloperonospora arabidopsidis.
(B) Core proteome differences between S. parasitica and Phy-
tophthora. Core protein sequences from P. sojae (green) and S.
parasitica (orange) are ordered by their amino acid similarity to
orthologous P. infestans proteins. Sequences with high similarity
(.50%) are shown in solid, while those with less similarity
(between 50% and 30%) are shown with a line. Sequences with
less than 30% are not shown.
Phytophthora. (A) Mobile elements in S. parasitica and Phytophthora.
The average copy number in P. infestans, P. sojae and P. ramorum is
used as the copy number for Phytophthora. The elements are sorted
based on the estimated copy number. (B) The S. parasitica element
LTR-Sp1 is similar to the Copia-like family (Q572G9_PHYIN) in
Phytophthora species. (C) The S. parasitica line element Line-Sp1
shows most homology with LINE elements found in fish and
amphibian species (no other similar elements were found in other
animal species). SwissProt protein species codes were used to name
the sequences. The phylogenetic tree was constructed by using the
neighbor joining method with 5000 replicates for bootstrap
Mobile element comparison between S. parasitica and
distribution of S. parasitica kinases compared to other organisms.
The kinases are named after the Standard Kinase Classification
Scheme at kinase.com. TK=tyrosine kinase; TLK=TK-like;
STE=STE7,11,20 family of MAP kinases; CMGC=(CDK,
MAPK, GSK3 and CLK) family; CK1=cell (casein) kinase 1
family; CAMK=Calmodulin/Calcium modulated kinase family;
AGC=Protein Kinase A, G, and C families. The unclassified
kinases are indicated in black. (B) S. parasitica contains a large
number of protein kinases that contain trans-membrane helices.
Pies are scaled to the total number of kinases in each species. (C)
Kinase genes that are induced in the germinating cyst stage in S.
parasitica compared to mycelia. Transcripts elevated more than
four-fold relative to vegetative stages are considered to be induced.
The expanded kinome of S. parasitica. (A) The
PLC1 and PLCs from various organisms. For phylogenetic
analysis, the PLCYc domains were determined by Smart
(http://smart.embl-heidelberg.de), alignments were made and
regions containing gaps were eliminated resulting in a total of 88
positions in the final dataset. The optimal tree was inferred using
the Neighbor-Joining method with 5000 replicates and construct-
ed using MEGA version 4. PLC sequences were derived from
NCBI (*), JGI databases (http://genome.jgi-psf.org/,#), the
Sanger Institute (http://www.genedb.org,@),
(Q39032*), AtPLC2 (Q39033*), AtPLC3 (Q56W08*), AtPLC4
(Q944C1*), AtPLC5 (Q944C2*), AtPLC6 (UPI000034EE4D*),
AtPLC7 (Q9LY51*), AtPLC8 (Q9STZ3*), AtPLC9 (Q6NMA7*);
Aureococcus anophagefferens (Auran; 18506#); Ciona intestinalis (Cioin;
XP_002129990*); Cryptosporidium parvum (Crypa; Q5CR08*); Danio
Esi0000_0131$); Emiliania huxleyi (209393#); Fragilariopsis cylindrus
(Fracy;186252#); Homo sapiens (as described by *); Naegleria
gruberi (Naegr; 1225#); Paralichthys olivaceus (Parol; ACA05829*);
Paramecium tetraurelia, (PLC1, see *); Phaeodactylum tricornutum
(Phatr; 42683#), Plasmodium falciparum (Plasmo; PF10_0132@);
(A) Phylogram of PLCYc domains of S. parasitica
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Salmo salar (Salsa; NP_001167177*); S. parasitica: Sap-PLC
Toxoplasma gondii (Toxgo; XP_002367229*). (B) Gene structure of
PLC genes. PLC is missing from other sequenced oomycete
genomes, but present in S. parasitica. Multiple introns have been
identified in the S. parasitica PLC gene.
(A) The pathway from acetyl-CoA to lanosterol. (B) The pathway
from lanosterol to zymosterol. The red box shows CYP51 sterol
demethylase, a target of azole anti-fungal chemicals. (C) Pathways
from zymosterol to cholesterol and fucosterol.
Sterol biosynthetic pathway inferred in S. parasitica.
cules. (A) Classes of infection-related molecules. Two groups of
PAMPs, elicitin-like and cys-rich-family-3 proteins are present in
both animal- and plant-pathogenic oomycetes (colored red). The
gray dots indicate infrequent occurrences. (B) Elicitin-like proteins
in S. parasitica and Phytophthora. The canonical Phytophthora and
Pythium elicitins are colored green. S. parasitica elicitin-like proteins
are divergent and form species-specific clades.
Phylogenetic distributions of infection-related mole-
of SNP content across 5 kb regions for Saprolegnia CBS and N12
strains. (B) Density of SNPs according to 5 kb regions of the CBS
genome. The mode for the SNP rate is 2.6%. The bulge on the left
side of the distribution likely corresponds to 5 kb regions of the
assembly that are mosaic between haplotype and consensus
diploid, as can be seen having overlap in the distribution shown
in the contour plot (Figure S1C). (C) Distribution of rates of
polymorphisms between strains CBS and N12. Both heterozygous
and homozogous polymorphic sites were considered across 5 kb
regions of the CBS genome with Illumina reads aligned from
strain N12. The mode for the %SNP was computed as 3.1%. (D)
Distribution of rates of polymorphisms within strain N12. Only
heterozygous sites were examined in the alignments of Illumina
N12 reads to the CBS strain’s genome. The mode for the %SNP
was computed to be 1.7%.
Distribution of rates of polymorphisms. (A) Summary
strain CBS223.65 and N12. Asterisks indicate significant differ-
ences between the gene family and the core orthologs (* p,0.001;
** p,1025) based on a non-parametric Z-test. Phytophthora data is
based on the published results of Raffaele et al. (2010). GSR (Gene
Sparse Region), GDR (Gene Dense Region). (B) Nucleotide
substitution rate between the separated haplotypes of the strain of
S. parasitica strain CBS223.65.
(A) Nucleotide substitution rate between S. parasitica
fish cells in vitro. (A) Amino acid sequence of fusion protein
SPRG_14052_mRFP-His6. The CRxxxxxCDxxExC disintegrin
motif is shaded in red. The mRFP sequence is indicated in blue,
the His-tag is in green. (B) RTG-2 cells were exposed to 3 mM of
mRFP, SpHtp1 or SPRG_14052_mRFP-His6and incubated for
30 min, before photography.
Predicted disintegrin SPRG_14052 does not enter
in the total gene set. (A) Genes differentially expressed during fish
cell interaction. (B) Differentially expressed genes in different life
stages. (C) The correlation coefficients of pairwise comparisons
between RNA-Seq data sets (p,0.001). (D) Transcript levels of a
Stage specific gene expression detected by RNA-Seq
subset of disintegrin-encoding genes in various life stages of S.
parasitica determined by RNAseq and qPCR. For RNAseq, the
log2 value of RKPM of a gene is plotted. For qPCR, transcript
levels are relative to the transcript levels of SpHtp1 in cysts and
normalized against the reference gene SpTub-b encoding for
tubulin. Error bars correspond to four biological replicaties.
enzymes and signaling enzymes, sterol metabolism, disintegrin-like
proteins and supplementary methods.
Supplementary information on: phospholipid modifying
Saprolegnia parasitica genome assembly statistics.
based on coverage and polymorphisms.
Assembled S. parasitica genome partitioned into classes
S. parasitica genes and SNPs percentages.
Chitin biosynthesis, modification and degradation in
enzymes in oomycetes.
Gene encoding nitrogen and sulphur assimilation
Saprolegnia parasitica and other oomycetes.
Phospholipid modifying and signaling enzymes in
Unique and expanded domains in Saprolegnia parasitica
Saprolegnia parasitica secretome.
Protein domain combinations in Saprolegnia parasitica.
Candidate effectors in Saprolegnia parasitica.
Polymorphism statistics for Saprolegenia strains.
germinating cyst stages as compared to the mycelial tissue by
Genes that are significantly induced in cyst and
Iinduced in cysts or germinating cysts stage.
Genes encoding lineage-expanded domains that are
Expression of peptidase genes in S. parasitica by RNA-
We thank members of the 2010 Saprolegnia Annotation Jamboree for
annotations, and Shiv Kale for useful discussions. We thank the Broad
Genomics Platform for their contribution and support. We thank James
Bochicchio for project management and Jonathan Goldberg, Sharvari
Gujja and Lucia Alvarado-Balderrama for genome annotation and data
The Genome Sequence of S. parasitica
PLOS Genetics | www.plosgenetics.org18 June 2013 | Volume 9 | Issue 6 | e1003272
Conceived and designed the experiments: RHYJ IdB BJH CJS JD-U CR
BMT PvW VB ER SMD-M. Performed the experiments: RHYJ IdB BJH
RB LL JC JZL CJS SW SKY QZ CR BMT PvW ER SMD-M AJP.
Analyzed the data: RHYJ IdB BJH RB LL JC GvdA AB BD LF EG FG
LJG-B NRH JZL MM HJGM PM CN SO DvR MS CJS MFS BS JHMS
SS ST HvdB JCV-A SW SKY QZ JD-U CR BMT PvW VB ER SMD-M.
Contributed reagents/materials/analysis tools: CN CR BMT PvW ER
SMD-M AJP. Wrote the paper: RHYJ IdB BJH RB LL FG CN CR BMT
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