BACKGROUND
the phytopathogenic oomycete phytophthora cactorum  j. schröt is capable of infecting an extremely wide range of hosts that span several plant families.
it often causes root, collar, and crown rots, as well as foliar and fruit infections.
it limits production for many economically important crops worldwide such as strawberry, apple, pear and rhododendron  <cit> .
additionally, p. cactorum is homothallic and produces sexual oospores that can survive for many years in the soil, which makes it more difficult to control the pathogen  <cit> .
at present, there is no effective chemical or cultural strategy available to limit diseases when environmental conditions become warm and wet.
as with many phytophthora pathogens, this species is especially problematic in low-lying or wet field conditions because infection often occurs through the release from sporangia of motile, flagellate and wall-less zoospores that can swim chemotactically or electrotactically toward host plants  <cit> .
once docking at the potential infection sites, zoospores encyst after shedding their flagella and bind to plant surfaces by means of adhesive glycoproteins  <cit> .
cysts germinate through extending a germ tube to penetrate the plant surface by enzymatic and mechanical means  <cit> .
subsequently, host colonization occurs through the growth of a coenocytic mycelium  <cit> .
the various steps of the life cycle of phytophthora have been studied at the cytological level  <cit> .
also molecular studies have demonstrated that these different stages require specific expression of many genes .
these life cycle stages of the pathogens are likely to be rich in molecules involved in establishment of infection and elicitation of plant defenses.
hence, the unraveling of the molecular processes regulating the life cycle stages of phytophthora is important to identify determinants of pathogenesis and improve control strategies.
recent molecular studies of phytophthora pathogens have focused on secreted effectors because of increasing evidence that these diverse proteins manipulate the defense responses of host plants  <cit> .
the genomes of several phytophthora species have been sequenced, including p. sojae, p. ramorum, p. infestans and p. capsici.
the sequencing and analyses of the genomes of other phytophthora pathogens such as p. parasitica, p. cinnamomi are currently being performed .
transcriptome studies using different approaches such as rna-seq and microarrays have been conducted to understand the pathogens and/or the interaction with their hosts .
all of this represents a rich trove of information on the effector repertoires of these pathogens.
various types of secreted effectors are predicted to act in the apoplast  or cytoplasm   <cit> .
apoplastic effectors, including elicitins, nlps , pcf-like proteins, cbel  proteins and enzyme inhibitors, are located at the interface between pathogens and hosts and execute functions outside of the host cell  <cit> .
elicitins, one type of pathogen-associated molecular patterns , can trigger plant cell death  response, normally known as hypersensitive reaction .
these extracellular proteins share a 98-amino-acid domain with a core of six conserved cysteines in the c-terminal domain  <cit> .
nlps can cause pcd in dicotyledonous plants and are identified by the presence of a common npp <dig> domain  <cit> .
the pcf-like toxin family represents one group of small cysteine-rich proteins from phytophthora species and is thought to have a toxic effect on plants  <cit> .
it has been originally named after the protein effector pcf , a  <dig> amino acid phytotoxic necrosis-inducing protein  <cit> .
cytoplasmic effectors are able to translocate inside host cells where they interfere with the host physiological functions including defense responses  <cit> .
one family of cell-entering effector proteins  shares an n-terminal rxlr amino acid motif   <cit> .
this motif is believed to assist in the translocation of the proteins into the host’s cytoplasm where the effectors suppress basal host resistance or activate effector-triggered immunity  depending on the host genotype  <cit> .
host translocation may also occur with variations of the rxlr motif  .
however, how such effectors cross several biological interfaces to reach the host cytoplasm remains an unclear and debated area of oomycete research  <cit> .
nevertheless, the importance of these proteins is underlined by the finding that all oomycete avirulence genes identified to date encode rxlr effectors .
due to selection pressure from the hosts, the avirulence effectors show extensive variations, including amino acid changes indicative of strong positive selection, gene truncations and deletions, and transcriptional silencing  <cit> .
the effector activity resides in the c-terminal regions of rxlr effectors  that are typically under positive selection  <cit> .
despite the extensive sequence diversity, the c-terminal regions of about half of rxlr effectors display a conserved core α-helical fold  that probably tolerates considerable plasticity  <cit> .
in addition to the rxlr effectors, other cytoplasmically-localized effectors have been identified in phytophthora species  <cit> .
the crinkler  protein family produces a leaf crinkling and necrosis phenotype when expressed in nicotiana benthamiana <cit> .
recent studies revealed that some crn effectors target host nuclei  <cit> .
a possible role for some crns towards phytophthora virulence has been suggested  <cit> .
intriguingly, a substantial number of phosphorylated crns were recently found in six life stages of p. infestans, from hyphae to appressoria, implying that some members of crn family could have functions other than to serve as effectors during infection  <cit> .
although the effector repertoire is generally highly divergent between species, features such as motifs or domains  <cit>  shared by effectors in different phytophthora species allow rapid identification of effector candidates from genome sequences.
despite its economic importance, molecular studies of p. cactorum have significantly lagged behind studies on other phytophthora species.
studies of the molecular basis of the pathogenicity of p. cactorum are limited to the chemical identification of α-elicitin  <cit>  and the phytotoxic protein pcf  <cit> .
changes in p. cactorum gene expression profiles prior to and during strawberry infection were recently studied using two approaches   <cit> .
a set of differentially expressed genes including  <dig> rxlr candidate genes was identified, but no further verification or functional characterization were conducted.
recent advancements in sequencing technologies have led to an explosive growth in the analysis of infection-related genes of oomycete plant pathogens .
the application of next generation sequencing techniques in non-model species promises to collect large amount of data necessary for functional studies rapidly and cost-effectively.
in order to gain more insights into the molecular mechanisms of p. cactorum pathogenicity, in the present study we report on the illumina sequencing, de novo assembly, annotation and analysis of the p. cactorum transcriptome in important life cycle stages.
a large number of genes and gene families were identified using different methods.
we investigated the potential effector arsenal including rxlr effectors.
the rxlr candidate effector dataset was searched for the conserved structure unit, wy-domain fold.
transcript levels of potential effector genes were monitored during the developmental and host infection stages of p. cactorum.
heterologous expression revealed that some effectors could elicit pcd.
sequence polymorphisms and positively selected sites in rxlr effector candidates were also analyzed.
in total, the work described herein provides a crucial foundation for further dissection of genes relevant to virulence in this broad-host-range phytopathogen.
RESULTS
sampling of p. cactorumlife cycle stages
to gain a global overview of the p. cactorum transcriptome and gene activity during the important life stages, we prepared a mixed cdna sample using rna from  <dig> different life stages: mycelia , sporangia , swimming zoospores , cysts  and germinating cysts with germ tubes  .figure  <dig>
life cycle stages of
p. cactorum
including cysts germinating under different conditions.
five successive life cycle stages are shown:  mycelia,  sporangia,  released sporangia and zoospores,  cysts, - germinating cysts.
in panel , the arrow indicates a zoospore being released from the sporangium.
cysts were germinated on a cellophane membrane placed on the top of an n. benthamiana leaf , or directly on n. benthamiana leaves , or in water .
in panel , the cysts were observed under a cryoscanning electron microscope .
the other cell types were observed using an olympus system microscope bx <dig>  photos were taken at  <dig> ,  <dig>  and  <dig>  min post-inoculation.
scale bars: , , 10 μm; , , , 50 μm; , 100 μm.
in this study, we found that p. cactorum can infect the model plant n. benthamiana at least through foliage and roots .
the infection by p. cactorum reproducibly caused visible leaf cell death, observed at  <dig>   <dig>   <dig>  and 24 h post-inoculation  using trypan blue staining .
in order to investigate early infection events by p. cactorum but to avoid the involvement of plant tissue in the pathogen rna samples for later transcriptome sequencing, a cellophane membrane placed on the n. benthamiana leaf was used as a surface for the induction of cyst germination as previously described for magnaporthe grisea <cit> .
the cysts germinating on both cellophane membranes  and n. benthamiana leaves  were morphologically similar, in contrast to cysts germinating in water .figure  <dig>
disease progressions in
n. benthamiana
caused by
p. cactorum
isolate  <dig>  leaves  or roots  were inoculated with a p. cactorum zoospore suspension.
trypan blue-stained leaves at  <dig>   <dig>   <dig> and 24 h post-inoculation .
on each leaf, the left side of the main vein was mock-inoculated with water while the right side was inoculated with zoospores.
plants were inoculated by pouring zoospore suspension around the roots.
upper panel, disease development on whole plants  <dig>   <dig>   <dig>   <dig>  5 days post-inoculation .
lower panel, root from plants in upper panel experiments were cut open to observe symptoms inside the roots.
ctrl, roots from control plants mock-inoculated with water.
to further test if the cysts germinating under this artificial condition display similar gene expression pattern to that of natural infection,  <dig> rxlr candidate genes  were selected for comparison with normalized read counts and real-time quantitative rt-pcr .
during the transcriptome sequencing period, illumina rna-seq was also utilized to perform massively parallel sequencing of p. cactorum my, zo and gc in our lab .
about  <dig> million sequence reads for each library  were mapped to the assembled reference transcriptome , and the transcript level of each gene in different libraries was calculated and normalized to the reads per kilobase of exon model per million mapped reads   <cit>  for comparison.
at the same time, expression of these rxlr candidate genes’ mrna was measured using qrt-pcr under the artificial condition.
a parallel qrt-pcr analysis was also performed with the cysts germinating directly on n. benthamiana leaves for comparison.
in gc versus my, all these  <dig> rxlr candidate genes showed the same expression pattern  under artificial condition as during natural infection .
this direct comparison faithfully demonstrated that genes differentially expressed in cysts germinating on cellophane show the same expression pattern as during natural infection, relative to the mycelium.
these results convinced us that cellophane membrane was a valid model for the cyst germination.
we did not harvest cysts germinating in water because in the absence of the leaf or membrane surface, cyst germination was significantly slower and non-uniform .
hence, rna from the cysts germinating on cellophane membrane was extracted at 60 min post inoculation and mixed equally with the my, sp, zo and cy rna samples for illumina rna-seq.
transcriptome sequencing and de novoassembly
we used illumina platform to perform p. cactorum transcriptome sequencing.
two biological replicates were generated and sequenced for mixed samples of five life cycle stages.
each sequenced sample yielded 2 ×  <dig> or 2 × 83-bp independent reads from either end of a cdna fragment .
after filtration of low-quality and adapter sequences, a total of  <dig> , <dig> cleaned cdna reads  was obtained and combined for analyses hereafter.
the percentage of q <dig> bases for the clean reads were >94%.
an average “g + c” content of above 56% was observed for the p. cactorum cdna sequences .
an overview of the assembled contigs, scaffolds and unigenes is presented in table  <dig>  initially, the reads were assembled into  <dig>  contigs.
after paired-end joining and gap-filling, the contigs were further assembled into  <dig>  scaffolds totaling  <dig> , <dig> bases.
the final clustering of scaffolds produced  <dig>  unique genes .
more than half of the unigenes  were longer than 500 bp and  <dig>  unigenes  were longer than  <dig> kbp.
the distribution of scaffolds and unigenes is shown in additional file  <dig>  for the randomly fragmented transcriptome, there was a positive relationship between the length of a given unigene and the number of reads assembled into it .
to evaluate the completeness of the transcriptome assembly, we used the core eukaryotic genes mapping approach  pipeline  <cit> , with the cegma subset of the  <dig> widely conserved core eukaryotic genes that are likely to have low frequencies of gene family expansion .
using this tool,  <dig>  of the  <dig> cegma genes mapped against the p. cactorum transcriptome assembly were identified.
the high recovery of cegma genes suggests a high quality transcriptome assembly  <cit> .table  <dig>
summary of transcriptome assembly for
p. cactorum
sequence annotation
the unigenes were annotated by aligning them with the deposited ones in  <dig> diverse databases including p. infestans, p. sojae, pythium, ncbi nr, ncbi nt, swiss-prot, trembl, interproscan databases .
the best one was selected from the matches with an e-value of less than 10− <dig>  in total,  <dig>   unigenes could be matched to the genes from at least one of  <dig> public databases .
the results demonstrated that of the  <dig>  unigenes,  <dig>   had significant amino acid sequence matches in the p. infestans database and  <dig>   retrieved significant hits in the p. sojae database while only  <dig>   could be mapped to pythium genes.
in contrast,  <dig>   of unigenes could be matched to the sequences deposited in the ncbi nr database.table  <dig>
summary of functional annotation of the
p. cactorum
transcriptome
to better define conserved genes in p. cactorum, protein sequences from the transcriptome assembly of this species  and three completely sequenced phytophthora pathogens, p. infestans , p. sojae  and p. ramorum , were clustered into gene families based on reciprocal pairwise sequence similarities .
in total,  <dig>  protein sequences from these four phytophthora species were clustered into  <dig>  groups.
of them,  <dig>  orthologous groups were shared by all four species.
a total of  <dig>  orthologous groups for p. cactorum,  <dig>  for p. infestans,  <dig>  for p. sojae, and  <dig>  for p. ramorum were generated.
about 12%  of p. cactorum putative proteins did not have orthologues in the other three phytophthora species, of which  <dig> protein sequences were clustered into  <dig> paralogous groups within the species .
the remaining predicted proteins  are considered singletons because they do not cluster at all in this analysis.figure  <dig>
four-way-venn-diagram of the distribution of unique and shared gene families among
phytophthora
species.
homologous proteins in p. cactorum, p. infestans, p. sojae and p. ramorum were clustered into gene families using tribemcl.
numbers in individual sections indicate number of gene families .
overlapping regions denote groups with at least two proteins of all species that are part of the intersection.
the first number under the organism name gives the total number of clusters that the respective organism contributes to; the second number describes the total number of each organism’s gene models/sequences that go into clusters.
the difference of the second number to an organism’s total input proteins gives the number of singletons .
by  <dig> march  <dig>  there had been  <dig> p. cactorum nucleotide sequences including  <dig> expressed sequence tags  deposited in the ncbi nt database.
in order to characterize these sequences, we queried them against our transcriptome assembly.
blastn comparisons revealed that  <dig> of  <dig>  nucleotide sequences excluding ests were mapped to  <dig> p. cactorum unigenes .
of  <dig> mapped sequences,  <dig>  were matched to the same unigene u <dig>  .
in contrast,  <dig> of  <dig>  ests were mapped to  <dig> unigenes .
out of them, some ests may be derived from the same gene and  <dig> redundant ests were mapped to  <dig> unigenes.
six unknown ests turned out to be clustered into ribosomal rna unigene u <dig> .
go annotation in terms of “biological process”, “molecular function” and “cellular component” was conducted for the annotated p. cactorum unigenes and, for comparison,  <dig>  gene models of p. infestans.
based on sequence similarity,  <dig>  p. cactorum unigenes could be categorized into  <dig> functional groups.
as shown in additional file  <dig>  the distribution pattern of p. cactorum sequences in different go categories  was quite similar to that of  <dig>  p. infestans genes.
the unigenes were assigned with one or more go terms.
for “cellular component”, “cell & cell part”, “organelle” and “membrane” terms are dominant.
for “molecular function”, genes related to “binding”  and “catalytic activity”  are highly represented.
regarding “biological process”, “metabolic process” and “cellular process” are the highly represented go terms.
we found a few genes from terms of “antioxidant activity” , “signaling” , and “localization” .
besides go analysis, the unigenes were subjected to a search against the cog database for functional prediction and classification.
in total,  <dig>  of the  <dig>  sequences retrieving a hit from the ncbi nr database could be assigned to cog classifications .
cog-annotated putative proteins were functionally classified into  <dig> protein families involved in cellular structure, biochemical metabolism, molecular processing, signal transduction and so on .
the cluster for “general function prediction” represents the largest unknown group , followed by “replication, recombination and repair” , “transcription”  and “signal transduction mechanisms” .
the following categories: “extracellular structures” , “nuclear structure”  and “cell motility” , represent the smallest groups.
in addition,  <dig> unigenes were assigned to “defense mechanisms” and  <dig> unigenes were assigned to “translation, ribosomal structure and biogenesis” .
to identify the biochemical pathways that are active in p. cactorum, we mapped the  <dig>  annotated sequences to the reference canonical pathways in kegg.
this is a complementary approach to categorize genes functions with the emphasis on biochemical pathways.
the process predicted a total of  <dig> pathways represented by a total of  <dig>  unigenes.
a summary of the sequences involved in these pathways is included in additional file  <dig>  these predicted pathways represented the majority of biochemical pathways for compound biosynthesis, degradation, utilization, and assimilation, and pathways involved in the processes of detoxification and generation of precursor metabolites and energy.
the pathways with most representation by unique sequences were “ribosome” , “chromosome”  and “spliceosome” .
searching and validation of the in silico-predicted p. cactorumeffector genes
like other plant oomycete pathogens, p. cactorum presumably secretes a battery of virulence proteins to promote infection.
the secretome for pathogenicity-related proteins was identified by comparison of p. cactorum unigenes against public databases.
a total of  <dig> effector genes with known or putative roles in virulence were identified .
notable families of secreted proteins include nlps, cbels with carbohydrate binding domains, elicitins and elicitin-like proteins, pcf-like proteins, and protease inhibitors  .
our data indicate that p. cactorum exhibits the same kinds of pathogenicity-related effector proteins that have been found in other oomycetes .table  <dig>
gene families potentially implicated in plant pathogenesis in
p. cactorum
p. cactorum
a
p. infestans
b
p. sojae
b
p. ramorum
b
pythium ultimum
b
hyaloperonospora arabidopsidis
b
albugo laibachii
b
saprolegnia parasitica
b
athe number of “real” effector genes in our dataset is yet to be determined though obvious pseudogenes are omitted.
bdata from other oomycete species are from haas et al.
<cit>  for p. infestans, tyler et al.
<cit>  for p. sojae and p. ramorum, lévesque et al.
<cit>  for pythium ultimum, baxter et al.
<cit>  for h. arabidopsidis, kemen et al.
<cit>  for a. laibachii, or jiang et al.
<cit>  for s. parasitica.
counts of annotated pseudogenes are omitted.
recently, many studies have shown a vast repertoire of apoplastic and cytoplasmic effector proteins including rxlr and crinkler  families in oomycetes .
the p. cactorum unigene dataset was searched for similarity to known oomycete effectors.
this search identified  <dig> potential rxlr effectors containing an rxlr or rxlr variant motif located downstream of the signal peptide.
to find the potential rxlr effectors without similarity to hits in sequenced genomes of oomycete species, all putative orfs from the assembly were generated and translated to amino acid sequences from both strands.
the predicted peptides were analyzed using the rxlr effector identification method  <cit> .
this yielded the same rxlr effector dataset as blastx comparison did.
another search for the amino acid motif rxlr was performed on these predicted peptides.
hmmer searches employing models described previously  <cit>  yielded five additional potential rxlr effectors.
signal peptide analysis was conducted using signalp  <dig>  and four candidates were excluded because of low signalp hmm probability .
these led to the final set of  <dig> unique potential rxlr effectors .
phylogenetic analysis of the phytophthora genus has shown that p. cactorum lies with p. infestans and p. parasitica in clade  <dig>  <cit> .
comparison of our rxlr dataset against p. parasitica inra- <dig>  genome was also performed to double check the effector searching .
we mapped a total of  <dig> effector candidates to p. parasitica rxlr genes, leaving  <dig> that matched to hypothetical protein-encoding genes and one not mapped to p. parasitica.
in contrast,  <dig> mapped to p. infestans rxlr genes and only  <dig> mapped to uncharacterized p. infestans genes  or no hits  .
four candidates matched to uncharacterized genes or no hit of both genomes at the same time.
those uncharacterized hit proteins were identified as not rxlr effectors because of absence of rxlr motif in the sequences.
among the  <dig> potential effectors,  <dig> contained a conserved rxlr motif.
ten contained a variant of rxlr motif  that is similar to the pexel translocation motif of plasmodium falciparum effectors .
the details of  <dig> candidates containing rxlr and/or deer motif or variants are provided in table  <dig>  these candidates include  <dig> intact orfs and  <dig> incomplete sequences.
the remaining  <dig> rxlr effector candidates are found not to contain either rxlr or deer motifs due to short sequences .table  <dig>
canonical and variant rxlr-deer motifs and the wy-domain found in
p. cactorum
transcriptome protein models
a
b
b
b
c
cd
e
cf
f
f
e
-value
f
f
pcrxlr1
pcrxlr2
pcrxlr3
pcrxlr4
pcrxlr5
pcrxlr6
pcrxlr7
pcrxlr8
pcrxlr9
pcrxlr10
pcrxlr11
pcrxlr12
pcrxlr13
pcrxlr14
pcrxlr15
pcrxlr16
pcrxlr17
pcrxlr18
pcrxlr19
pcrxlr20
pcrxlr21
pcrxlr22
pcrxlr23
pcrxlr24
pcrxlr25
pcrxlr26
pcrxlr27
pcrxlr28
pcrxlr29
pcrxlr30
pcrxlr31
pcrxlr32
pcrxlr33
pcrxlr34
pcrxlr35
pcrxlr36
pcrxlr37
pcrxlr38
pcrxlr39
pcrxlr40
pcrxlr41
pcrxlr42
pcrxlr43
pcrxlr44
pcrxlr45
pcrxlr46
pcrxlr47
pcrxlr48
pcrxlr49
pcrxlr50
pcrxlr51
pcrxlr52
pcrxlr53
pcrxlr54
pcrxlr55
pcrxlr56
pcrxlr57
pcrxlr58
pcrxlr59
pcrxlr60
pcrxlr61
pcrxlr62
pcrxlr63
pcrxlr64
aeffectors were predicted from the unigene sequences.
bhidden markov model  probability, nn mean s score, and signal peptide length were predicted using signalp v <dig> .
call sequence coordinates are given in amino acid residues.
ddetermined as two or more d,e,n and/or q residues followed by a r or k residue: { <dig> }.
eevidence codes for rxlr gene prediction: m , b , p , and r .
fwy-domain was predicted using the hmm for the wy-domain as described in  <cit>  with the hmm score cut-off of  <dig> .
the “—” means that no wy-domain was predicted.
we performed similar searches to identify crn effectors in p. cactorum.
in total,  <dig> potential crn proteins were identified .
three other pccrns were not picked up due to too short orf   <cit>  or lacked the characteristic n-terminal ~50-amino-acid lflak motif  <cit> .
the majority  of coding sequences lacked both 5′ and 3′ sequences.
twenty lacked 5′ sequences while four lacked a stop codon and apparently lacked 3′ sequences.
three sequences were complete, but only one  were identified with a predicted signal peptide .
pccrn <dig> was previously detected in a transcriptional study of p. cactorum  where it was found up-regulated during cyst germination and host infection  <cit> .
the number of “real” effector genes in our dataset is yet to be determined although obvious pseudogenes were omitted.
however, some of the predicted effector genes were validated in this study.
twenty-three effector sequences including  <dig> rxlrs,  <dig> nlps and one pcf-like were selected to check the accuracy of the computational analysis .
they were tested by pcr amplification using p. cactorum genomic dna.
a total of  <dig> amplifications produced a unique band of the expected size .
one sequence, pcrxlr <dig>  produced two amplified bands, but the brighter one exhibited the expected size, suggesting the lower band resulted from non-specific amplification.
three primer pairs towards the rxlr candidate genes  did not produce expected bands .
these  <dig> genes were then full-length cloned and sequenced, yielding the same sequences as transcriptome assembly data.
due to the approximately 87% correct predictions , the in silico analysis in this study was considered as providing reliable results.figure  <dig>
pcr amplification of a subset of the  <dig> predicted
p. cactorum
unigenes.
pcr amplification was performed on p. cactorum isolate  <dig> genomic dna with  <dig> primer pairs, and the amplicons were electrophoresed on an agarose gel .
lane marker, dl- <dig> dna marker ladder .
gene names and categories are listed on the top of the gel picture.
the star indicates a non-specific amplification band for pcrxlr <dig>  lane water, water control with pcnlp <dig> primers .
the wy-domain is conserved in about half of p. cactorumrxlr effectors
recently, it was identified by structural biology that wy-domain is a structure unit conserved in many phytophthora and h. arabidopsidis rxlr effectors  <cit> .
we searched p. cactorum rxlr effector candidates using the hmm for the wy-domain as described previously  <cit> .
using the score cut-off of  <dig> , we revealed that  <dig> of  <dig>  of p. cactorum rxlr effector candidates contain wy-domain-like sequences .
wy-domains can also be found in tandem repeats, and we identified  <dig> candidate effectors with more than one wy-domain.
two candidate effectors, pcrxlr <dig> and pcrxlr <dig>  contain  <dig> wy-domains.
therefore, many p. cactorum rxlr effector candidates comprise either single or tandem arrays of wy-domains.
the expression of most effector genes is induced during p. cactoruminfection
our study showed that p. cactorum can infect the model plant n. benthamiana .
no evident macroscopic symptoms were observed in the root until  <dig> hpi .
the disease progressed quickly in the plants, and by  <dig> d post-inoculation all inoculated plants were wilting or dead .
to investigate the effector gene expression pattern changes during host infection, the infected n. benthamiana roots were harvested at  <dig> infection time-points .
this assured that the whole infection period of p. cactorum on n. benthamiana could be investigated.
cdna samples from four life stages  were also obtained as described previously but with one modification in which here gc was induced directly on n. benthamiana leaves.
hence gc sample used here is a mixture of microbe and plant materials.
expression experiments were performed for  <dig> effector genes including  <dig> rxlrs, one elicitin  and one nlp .
those  <dig> representative rxlr unigenes were chosen based on a consideration of the signal peptide confidence, the intact orf and rxlr-deer presence.
the transcript levels in the  <dig> rna samples were assessed using semi-quantitative rt-pcr.
eleven of the designed rxlr primer pairs produced unspecific  or no  fragments for the infection samples, and were not included for further analyses.
figure  <dig> shows the expression patterns obtained for the remaining  <dig> rxlr and  <dig> other genes.
all these rxlr genes were up-regulated during the host invasion, relative to my that can be regarded as the vegetative growth stage.
in particular, all but one rxlr gene  were highly expressed during the earliest infection stages .
of these  <dig> early expressed genes,  <dig> also showed induced expression at gc which is an essential pre-infection stage of p. cactorum.
the differential expression of  <dig> rxlr candidate genes  in comparison between gc and my was further validated by qrt-pcr and rpkm analyses .
two rxlr genes  were previously detected in a transcriptional study of p. cactorum  where they were expressed during strawberry infection and cyst germination  <cit> .
that result is consistent with the result obtained in this study.figure  <dig>
expression patterns of 19
p. cactorum
effector genes during life cycle and infection stages.
lanes: my, plate-grown mycelia; sp, sporangia; zo, zoospores; gc, germinating cysts directly on n. benthamiana;  <dig>  -  <dig>  n. benthamiana roots at  <dig> ,  <dig>   <dig>   <dig>   <dig>   <dig>  96 hours post-inoculation ; pl, mock-inoculated plants; w, control leaves inoculated with distilled water.
gene names and categories are listed on the left and the amplicon sizes are listed on the right.
all sizes were as expected, and the experiment was performed with at least three biological replicates.
in addition, the expression of two other effector genes was examined .
the elicitin gene pcell <dig> showed stronger expression both at my and during late infection of n. benthamiana  versus at  <dig>  hpi.
the nlp gene pcnlp <dig>  however, showed expression only at zo and  <dig> hpi.
p. cactorumeffectors elicit plant cell death and are conserved across oomycete species
expression studies indicated that we had identified infection-related effector genes in p. cactorum.
to begin an investigation of the activity of those potential effectors in planta, we conducted heterologous expression assays in n. benthamiana using infiltration of a. tumefaciens cells with the vector pgr <dig> .
these methods have been proved valuable in the characterization and determination of the function of a variety of effector genes .
all of the  <dig> aforementioned effectror genes were cloned into the transient expression vector pgr <dig>  a known elicitor of cell death, the p. infestans pamp inf <dig>  <cit> , was used as positive control.
a. tumefaciens strains harboring gfp served as negative control.
the elicitin pcell <dig> , nlp effector pcnlp <dig>  and four rxlr effectors   triggered leaf cell death in n. benthamiana as vigorously as inf <dig> .
expression of pcrxlr <dig>  pcell <dig> and pcnlp <dig> resulted in leaf chlorosis throughout the entire infiltration zone by  <dig> dpi, followed by initiation of cell death at  <dig> dpi.
by  <dig> dpi, the zone of infiltration was completely dehydrated.
the cell death symptoms triggered by pcrxlr <dig> and pcrxlr <dig> developed about  <dig> d after infiltration whereas the symptoms caused by pcrxlr <dig> developed about  <dig> d after infiltration.
in contrast, only mosaic symptoms due to pvx were observed following infiltration with the remaining  <dig> rxlr effector genes or with gfp under the same conditions.figure  <dig>
transient assays of
phytophthora
rxlr, nlp and elicitin effectors in
n. benthamiana
.
the p. cactorum elicitin pcell <dig> , nlp pcnlp <dig> , four rxlrs  , p. capsici rxlr pcaprxlr <dig>  and the alleles of pcrxlr <dig> and pcrxlr <dig>  triggered plant cell death.
the plant leaves were infiltrated with a. tumefaciens  cells to express each of these effectors.
agro-infiltration sites in each n. benthamiana leaf expressing inf <dig>  were the positive controls.
the other sites in leaves expressing gfp only, or infiltrated by 10 mm mgcl <dig> buffer only served as negative controls.
pcaprxlr <dig> is the ortholog gene of pcrxlr <dig> in p. capsici.
the alleles of pcrxlr <dig> in the isolates ph-1-14- <dig> and ph-1-8- <dig> are labeled as pcrxlr <dig>  and pcrxlr <dig> , respectively while the allele of pcrxlr <dig> in pc <dig> as pcrxlr <dig> .
photographs were taken  <dig>  or  <dig>  d after infiltration.
the circles indicated the agroinfiltration areas.
we performed multiple sequence alignment of these elicitors of cell death, i.e.
four rxlr effectors , pcell <dig> and pcnlp <dig> to determine their conservation within oomycetes .
based on the conserved features and domain organization of oomycete effectors, the presence of a canonical rxlr motif and an eer motif was identified at n-termini of p. cactorum rxlr effectors and their orthologs in other phytophthora species .
blastx results showed that p. cactorum pcrxlr <dig>  pcrxlr <dig>  pcrxlr <dig> and pcrxlr <dig> have 34%, 73%, 32% and 41% identity to p. infestans rxlr homologues, respectively.
in contrast, these four effectors displayed higher sequence identity  to p. parasitica homologues.
however, the p. parasitica homologues of pcrxlr <dig> and pcrxlr <dig> were annotated as hypothetical proteins in the inra- <dig>  reference genome  although the canonical rxlr-eer motif is present .
except pcrxlr <dig>  the other three rxlr effectors have homologues in p. capsici and p. sojae .
noticeably, pcrxlr <dig> and pcrxlr <dig> showed high sequence similarity  to p. sojae effectors avh <dig> and avh <dig>  respectively, which have been identified to trigger cell death in n. benthamiana and soybean  <cit> .
as reported previously  <cit> , the rxlr motif of pcrxlr <dig>  pcrxlr <dig>  pcrxlr <dig> and their orthologs in other phytophthora species is confined to position 30– <dig> amino acids .
however, analysis methods employing this constraint most likely eliminate genuine effectors.
in this study, the canonical rxlr motif is located between residues  <dig> and  <dig> in the amino acid sequences of pcrxlr <dig> and its orthologs .
data mining revealed that the coding sequence of its p. capsici ortholog, hereafter referred to as p. capsici rxlr protein  <dig> , is embedded in a cdna clone cbot37-c <dig>  but not annotated in the p. capsici reference genome .
we cloned pcaprxlr <dig> into pgr <dig> and performed transient assay as described previously.
intriguingly, pcaprxlr <dig> also triggered cell death in n. benthamiana leaves about  <dig> d after infiltration .
the p. capsici orthologs of pcrxlr <dig> and pcrxlr <dig> did not induce pcd in n. benthamiana.
the above results together suggest that these rxlr effector proteins from different phytophthora pathogen species trigger similar responses in plant families.
one nlp effector of interest, pcnlp <dig>  showed  <dig> of  <dig> amino acids conserved across four different phytophthora species and an additional oomycete, pythium ultimum, whereas  <dig> were shared with p. parasitica, corresponding to 85% identity .
the data showed that all aligned nlps contained two conserved cysteine residues which defined these proteins as type i nlp  <cit> .
we also noted that the central conserved ghrhdwe heptapeptide motif is present in all nlps .
the elicitin pcell <dig> shared  <dig> amino acids with four phytophthora species and py.
ultimum, whereas  <dig> of  <dig> amino acids  were conserved between p. cactorum and p. infestans .
polymorphic sites in p. cactorumrxlr effector sequences
the four rxlr genes that triggered pcd were examined in a collection of seven p. cactorum isolates , to determine whether sequence polymorphisms may exist in these genes.
effector genes may exhibit a high level of polymorphism if they are subject to diversifying selection as a result of pressure from plant resistance genes.
two  of the four rxlr genes were found to exhibit non-synonymous polymorphisms that altered the amino acid sequences they encoded .
variations in  <dig> amino acid residues were identified in pcrxlr <dig> among the  <dig> sequenced isolates .
these  <dig> residues were located at the c-terminus of the sequence.
the same fragment was not amplified from the isolate pc <dig> by pcr, suggesting that the gene may be deleted or substantially altered in this isolate.
noticeably, the effector sequence was identical among the three european strawberry isolates, and in the two persian walnut isolates.
pcrxlr <dig> could be amplified from all strains but fin <dig>  but showed only one polymorphic site at the n-terminus among those six isolates .
in contrast, pcrxlr <dig> and pcrxlr <dig> showed no amino acid polymorphisms among the  <dig> tested isolates.
the ability of the different effector alleles to trigger pcd was tested .
the alleles of pcrxlr <dig> amplified from the isolates ph-1-8- <dig> and ph-1-14- <dig> induced the same pcd as the one from the isolate  <dig> did .
similarly, pcrxlr <dig> allele in pc <dig> also produced the same symptom on plants as pcrxlr <dig> of the isolate  <dig> did .
the results indicated that the polymorphic sites in these rxlr effectors do not affect their ability to trigger plant cell death.figure  <dig>
sequence polymorphisms of rxlr effector alleles among
p. cactorum
isolates.
the protein sequences of pcrxlr <dig> in six isolates  were compared.
the allele was not obtained from the isolate pc <dig>   the protein sequences of pcrxlr <dig> in six isolates  were compared.
the allele was not obtained from the isolate fin <dig>  the putative rxlr and eer motifs of both effectors were denoted by red boxes.
their predicted signal peptide sequences were underlined.
the polymorphic sites were marked by blue asterisks for the visualization.
we further examined the strength of natural selection acting upon the effectors  by estimating the ratio of the non-synonymous substitution rate  to the synonymous substitution rate  .
we defined the boundary of the n  and c termini of protein sequences as the 70th  or 116th  amino acid .
the yn <dig> tool in the paml v <dig>  software package was used to calculate the ratio of dn to ds <cit> .
three representative sequences made up three pairwise comparisons for the test of pcrxlr <dig>  there is no polymorphism in the n-terminus of pcrxlr <dig> but in the c-terminus.
however, no significant evidence of selection pressure in the c-terminus of pcrxlr <dig> was identified though the pairs 10300/ph-1-8- <dig>  ph-1-8-03/ph-1-14- <dig> displayed dn > ds and the pair 10300/ph-1-14- <dig> showed dn < ds.
single polymorphism site was found in the n-terminus among alleles of pcrxlr <dig>  although dn > ds, it may not be significant .
consistent with these results, model comparisons using the computer program codeml  <cit>  did not reveal any positively selected sites for either of the rxlr effectors.table  <dig>
d
n
/d
s
analysis of pairwise comparisons of the rxlr alleles
b
b
n
s
n
/d
s
a
p
n
s
n
/d
s
a
p
n
s
n
/d
s
a
p
pcrxlr6
pcrxlr7
athe ‘∞’ in column dn/ds refers to pairwise comparisons in which dn > <dig> and ds = <dig>  and ‘–’ refers to pairwise comparisons in which dn = <dig> and ds =  <dig>
bthe n-terminus refers to the sequence from the 1st to the end of deer, and the c-terminus refers to the sequence from the immediate residue after deer to the end, except the stop codon.
discussion
despite the worldwide economic impact of diseases caused by p. cactorum, little is known about the molecular basis of the pathogenicity of this species.
the availability of the genome sequences from other pathogens and their host plants has greatly advanced our understanding of pathogen virulence and host resistance  <cit> .
davis and yu  <cit>  and shulaev et al.
<cit>  presented the first genetic map and a genome sequence, respectively, for woodland strawberry , which is one important host of p. cactorum.
in contrast, a lag still exists in developing these resources for the hemibiotrophic pathogen p. cactorum.
chen et al.
<cit>  provided a first overview of genes that may be involved in p. cactorum pathogenicity.
however, the sequence data obtained in that study were limited.
additionally, due to the short sequences and lack of a reference genome, cloning strategies such as rapid-amplification of cdna end  had to be employed to get the full length genes for ests of interest  <cit> .
the sequence data created in this study therefore represent a substantial expansion of the transcriptome resources available for p. cactorum and a glimpse into its pathogenicity mechanisms.
due to the common problem that contamination of plant material in infection-stage samples often makes it difficult to study the plant-microbe interactions  <cit> , examining phytophthora life cycle stages such as germinating cysts may be a useful alternative for investigating the transcriptional changes of infection-related phytophthora genes.
previous studies have shown that molecules involved in establishment of infection and elicitation of plant defenses are extensively expressed during the life cycle stages .
hence, in the present study the p. cactorum transcriptome sequences were generated by illumina sequencing from five successive life stages , and assembled by de novo short read.
to prepare the important pre-infection stage, the germinating cysts , a cellophane membrane was placed on n. benthamiana leaf to mimic natural infection and induce germination of cysts while minimizing contamination of plant material.
microscopic analysis first demonstrated that germinating cysts on cellophane morphologically resemble the ones germinating directly on n. benthamiana leaves, in contrast to the ones in water .
second, the gene expression pattern in cysts germinating under both mimicry and natural conditions was investigated and compared.
in gc versus my, the rxlr candidate genes  under mimic condition showed the same expression pattern as during natural infection .
the direct comparison provided further justification that our mimic experiments closely mirror the infection process occurred in fields.
thus, cellophane membrane was a valid model for the cyst germination.
the in vitro inoculation method could be applied to other similar plant-pathogen interaction studies.
using the cegma tool,  <dig>  of the  <dig> conserved single-copy eukaryotic genes mapped against the transcriptome assembly were identified in our study.
similarly,  <dig> % and 95% of  <dig> cegma genes were reportedly detected in a. laibachii and h. arabidopsidis genome assemblies, respectively  <cit> .
the high recovery of 98% of cegma genes suggests a high quality p. cactorum transcriptome assembly in the present study.
comparisons to sanger-derived sequences from p. cactorum , and pcr validation  further showed that the transcript assemblies are robust.
comparison of assembled gene models  to gene catalogs of other oomycete species by blastx analysis and functional annotation  indicate that the sequences represent an extensive catalog encompassing a large proportion of the genes expressed in p. cactorum.
in the study, we used the data from tribemcl to globally examine the conservation of p. cactorum genes across phytophthora species .
overall,  <dig>  gene families contained sequences from all four species, representing about 65%  of the total number of gene families identified in this study.
of that,  <dig> gene families were specific for p. cactorum and p. infestans.
in contrast,  <dig> gene families were specific for p. cactorum and p. sojae, and  <dig> for p. cactorum and p. ramorum.
this fits well with the fact that p. cactorum lies more closely with p. infestans than with others in the phylogenetic tree  <cit> .
among those  <dig>  putative p. cactorum proteins that lack homologs in other species,  <dig> were grouped into  <dig> paralogous groups .
the remaining  <dig>  were considered singletons, as they lack homologs within their own proteome or in the other taxa.
this number is a bit larger than the counterpart numbers from each of other three species .
this could be due to the sequencing depth and therefore assembly in this study.
the assembly quality of these sequences could be improved in the future by even deeper sequencing and by genome sequencing.
alternatively, these proteins could potentially represent p. cactorum-specific orphan genes.
however, more research is required to dissect this.
on the basis of gene annotations and pathway analyses, the p. cactorum unigenes are predominantly involved in cellular and metabolic processes, the binding and catalytic activities and cellular components .
similar results were found in p. infestans .
in addition, overall  <dig>  unique transcripts were predicted to be involved in  <dig> kegg metabolic pathways, with two major pathways  comprised of over  <dig> unigenes .
the predicted pathways together with the gene annotations will be useful for further investigations of gene function in the future.
potential apoplastic and host-translocated effectors could be predicted from the assembled transcript sequences.
in the present study,  <dig> p. cactorum genes were identified encoding transporter or effector proteins with putative roles in virulence , with a repertoire similar to other oomycete plant pathogens.
the p. cactorum transcripts obtained here represent the genes expressed during important life stages, and may therefore not represent all genes that are present in the genome.
the p. cactorum effector data set could be expanded by including additional life cycle and infection stages of p. cactorum.
sequencing of the genomes of phytophthora plant pathogens has revealed a variable number of rxlr effectors in different species  <cit> .
we identified  <dig> potential rxlr effectors in the p. cactorum transcriptome,  <dig> of which contained a conserved rxlr motif .
additionally,  <dig> potential effectors contained a variant of rxlr motif  that is similar to the pexel translocation motif of pl.
falciparum effectors .
plasmodium pexel domains can functionally replace the rxlr-deer region of avr1b from p. sojae and p. infestans avr3a  <cit> .
furthermore, mutation of the fourth position of the p. sojae rxlr motif did not affect translocation  <cit> .
of the  <dig> p. cactorum rxlr effector candidates,  <dig> sequences appeared to be complete .
we examined  <dig> representative effectors  using both rt-pcr  and pcr cloning .
seventeen full length sequences were cloned and confirmed to be rxlr genes.
the other  <dig> sequences were not further analyzed due to unspecific  or no  bands amplified from infection-stage samples in rt-pcr assays.
among these  <dig> sequences, only one can not produce any bands from all the test samples .
more specific primers towards these  <dig> sequences should be designed to dissect this.
and, more work is needed to determine the number of functional or “real” genes in this dataset though obvious pseudogenes were excluded.
two rxlr genes  out of  <dig> rxlr gene candidates reported in a previous study  <cit>   were re-detected in this study.
the others mostly discovered by effector-specific differential display  technique  <cit>  have not been re-detected in this study.
however, this is not surprising because effector genes could be differentially expressed at different stages in response to different conditions.
the  <dig> rxlr effector candidates were detected by rna-seq during the pathogen developmental stages in the present study while most  of  <dig> potential rxlr effectors were identified during the strawberry infection by esdd in the previous study  <cit> .
some effector genes could be stage-specific and therefore not detected in both cases.
the c-terminal regions of oomycete rxlr effectors carry the biochemical effector activity and about half of these proteins share a conserved wy-domain  <cit> .
this wy-domain fold may be an adaptive structural unit that can support effector diversification to gain new functions and/or evade plant host immunity  <cit> .
using hmm–based sequence searches, wy-domain had also been detected in our p. cactorum rxlr effector candidates, with  <dig> out of  <dig>  containing this fold  .
this strengthens that the wy-domain unit could be critical for the success of oomycete plant pathogens.
however, the reason why the wy-domain is preserved and its contribution to the functions of the effectors are still unknown.
future studies are required to help define the roles of the wy-domain fold in the virulence mechanisms of these pathogens.
we examined the transcript levels of those  <dig> representative effector genes by hemi-quantitative rt-pcr and observed high expression during the host infection for  <dig> of them .
nearly half of the rxlr genes were differentially expressed during the life cycle and early infection stages of the p. cactorum.
the expression patterns of the rxlr effector genes are consistent with their expected roles in assisting the colonization of plant tissues by manipulation of host defense responses  <cit> .
to explore their predicted roles further, we tested if the rxlr effectors could cause phenotypic reactions when expressed in host plants and found that four effectors could elicit plant cell death .
effectors from other oomycete plant pathogens, including avh <dig>  avh <dig>  avh <dig> from p. sojae, pcaprxlr <dig> from p. capsici , pscrxlr <dig> and pcqne from pseudoperonospora cubensis and members of the crn family from p. infestans and p. sojae, have also been shown to elicit similar phenotypes when transiently expressed in n. benthamiana <cit> .
the counter-intuitive ability of oomycete effectors that are expressed during biotrophy to trigger cell death has been inferred to reflect the ability of the effectors to manipulate the plant defense responses and/or the ability of the plant defense machinery to initiate effector-triggered immunity.
wang et al.
<cit>  showed that a minority of p. sojae rxlr effectors  triggered cell death in the host, soybean, or in n. benthamiana, and that a majority of tested p. sojae effectors could suppress the cell death triggered by those effectors.
thus during natural infection it is likely that such effectors do not actually induce cell death, but instead manipulate the plant immune system in other ways that promote infection.
previous studies have shown how the primary sequences of many rxlr effectors have been shaped by positive selection, and how the genes themselves undergo accelerated birth and death evolution  <cit> .
two of the cell death-inducing effectors, pcrxlr <dig> and pcrxlr <dig>  exhibited polymorphisms but no significant evidence of natural selection among seven p. cactorum isolates .
the polymorphisms were localized basically to the c-terminal effector domains.
generally the amino acid sequences of each of both rxlr effectors were identical within isolates from the same host while different among isolates from different hosts.
these host-related patterns may have been molded by host preferences, geographic isolation and/or degree of specialization, since the rxlr secretome is at the front line in the evolution of the host-pathogen interaction  <cit> .
the polymorphic nature of these p. cactorum rxlr effectors reinforces that these genes may play an important role in the microbe-plant interaction.
however, no residues under positive selection in these two rxlr effectors were identified.
this is possibly because only seven p. cactorum isolates were investigated for sequence polymorphism.
the alleles of pcrxlr <dig> and pcrxlr <dig> retain pcd-triggering activity although their sequences showed polymorphism.
the p. cactorum pcd-inducing rxlr effectors are highly conserved across several phytophthora species .
ectopic expression data showed that the p. capsici ortholog of pcrxlr <dig>   and p. sojae orthologs of pcrxlr <dig> and pcrxlr <dig>   <cit>  also triggered pcd in n. benthamiana, emphasizing their importance.
these pcd-inducing effectors maybe represent few rxlr effectors conserved across phytophthora species  <cit> .
whether such rxlr ortholog pair targets a common plant component needs further investigations.
in this study, the genes for the elicitin pcell <dig> and the nlp pcnlp <dig> were also validated, cloned and transiently expressed in plants.
both genes showed distinct expression during the developmental and infection stages .
ectopic expression in n. benthamiana displayed that both genes can trigger plant cell death .
recent studies on other phytophthora species revealed similar results  <cit> .
based on multiple sequence alignment, we found that these effectors are highly conserved across oomycete species .
together, these results suggested their important roles in the pathogenicity of p. cactorum.
future work should determine the number of functional or “real” genes encoding these effectors in p. cactorum and elucidate their functional roles in the pathogen’s virulence.
CONCLUSIONS
as a root pathogen, and because of its broad host range, p. cactorum is an attractive model for understanding phytophthora infection.
sequencing of the p. cactorum transcriptome has provided a wealth of information about the proteins expressed during the important life stages, especially the rxlr effectors.
this is the first publication using next generation sequencing technology for p. cactorum without genome information.
we have demonstrated the reliability of the transcriptome assembly by cegma, pcr validation and rt-pcr analyses.
a large number of genes including  <dig> rxlr effector candidates and gene families of p. cactorum were identified.
about half of the rxlr effector candidates are predicted to have a similar overall conformation, termed the wy-domain fold.
additionally, we have demonstrated the identification and characterization of pcd-triggering rxlr, nlp and elicitin effectors.
more studies will however be required to determine the roles of infection-related effector genes in the pathogenesis.
the results will ultimately help to elucidate the mechanisms underlying the pathogenicity of p. cactorum and to design effective control strategies for the diseases caused by this broad host range pathogen.

 the phytopathogenic oomycete phytophthora cactorum j. schröt is capable of infecting an extremely wide range of hosts that span several plant families. it often causes root, collar, and crown rots, as well as foliar and fruit infections. it limits production for many economically important crops worldwide such as strawberry, apple, pear and rhododendron. additionally, p. cactorum is homothallic and produces sexual oospores that can survive for many years in the soil, which makes it more difficult to control the pathogen. at present, there is no effective chemical or cultural strategy available to limit diseases when environmental conditions become warm and wet. as with many phytophthora pathogens, this species is especially problematic in low-lying or wet field conditions because infection often occurs through the release from sporangia of motile, flagellate and wall-less zoospores that can swim chemotactically or electrotactically toward host plants. once docking at the potential infection sites, zoospores encyst after shedding their flagella and bind to plant surfaces by means of adhesive glycoproteins. cysts germinate through extending a germ tube to penetrate the plant surface by enzymatic and mechanical means. subsequently, host colonization occurs through the growth of a coenocytic mycelium. the various steps of the life cycle of phytophthora have been studied at the cytological level. also molecular studies have demonstrated that these different stages require specific expression of many genes. these life cycle stages of the pathogens are likely to be rich in molecules involved in establishment of infection and elicitation of plant defenses. hence, the unraveling of the molecular processes regulating the life cycle stages of phytophthora is important to identify determinants of pathogenesis and improve control strategies. recent molecular studies of phytophthora pathogens have focused on secreted effectors because of increasing evidence that these diverse proteins manipulate the defense responses of host plants. the genomes of several phytophthora species have been sequenced, including p. sojae, p. ramorum, p. infestans and p. capsici. the sequencing and analyses of the genomes of other phytophthora pathogens such as p. parasitica, p. cinnamomi are currently being performed. transcriptome studies using different approaches such as rna-seq and microarrays have been conducted to understand the pathogens and/or the interaction with their hosts. all of this represents a rich trove of information on the effector repertoires of these pathogens. various types of secreted effectors are predicted to act in the apoplast or cytoplasm. apoplastic effectors, including elicitins, nlps, pcf-like proteins, cbel proteins and enzyme inhibitors, are located at the interface between pathogens and hosts and execute functions outside of the host cell. elicitins, one type of pathogen-associated molecular patterns, can trigger plant cell death response, normally known as hypersensitive reaction. these extracellular proteins share a 98-amino-acid domain with a core of six conserved cysteines in the c-terminal domain. nlps can cause pcd in dicotyledonous plants and are identified by the presence of a common npp domain. the pcf-like toxin family represents one group of small cysteine-rich proteins from phytophthora species and is thought to have a toxic effect on plants. it has been originally named after the protein effector pcf, a amino acid phytotoxic necrosis-inducing protein. cytoplasmic effectors are able to translocate inside host cells where they interfere with the host physiological functions including defense responses. one family of cell-entering effector proteins shares an n-terminal rxlr amino acid motif. this motif is believed to assist in the translocation of the proteins into the host’s cytoplasm where the effectors suppress basal host resistance or activate effector-triggered immunity depending on the host genotype. host translocation may also occur with variations of the rxlr motif. however, how such effectors cross several biological interfaces to reach the host cytoplasm remains an unclear and debated area of oomycete research. nevertheless, the importance of these proteins is underlined by the finding that all oomycete avirulence genes identified to date encode rxlr effectors. due to selection pressure from the hosts, the avirulence effectors show extensive variations, including amino acid changes indicative of strong positive selection, gene truncations and deletions, and transcriptional silencing. the effector activity resides in the c-terminal regions of rxlr effectors that are typically under positive selection. despite the extensive sequence diversity, the c-terminal regions of about half of rxlr effectors display a conserved core α-helical fold that probably tolerates considerable plasticity. in addition to the rxlr effectors, other cytoplasmically-localized effectors have been identified in phytophthora species. the crinkler protein family produces a leaf crinkling and necrosis phenotype when expressed in nicotiana benthamiana. recent studies revealed that some crn effectors target host nuclei. a possible role for some crns towards phytophthora virulence has been suggested. intriguingly, a substantial number of phosphorylated crns were recently found in six life stages of p. infestans, from hyphae to appressoria, implying that some members of crn family could have functions other than to serve as effectors during infection. although the effector repertoire is generally highly divergent between species, features such as motifs or domains shared by effectors in different phytophthora species allow rapid identification of effector candidates from genome sequences. despite its economic importance, molecular studies of p. cactorum have significantly lagged behind studies on other phytophthora species. studies of the molecular basis of the pathogenicity of p. cactorum are limited to the chemical identification of α-elicitin and the phytotoxic protein pcf. changes in p. cactorum gene expression profiles prior to and during strawberry infection were recently studied using two approaches. a set of differentially expressed genes including rxlr candidate genes was identified, but no further verification or functional characterization were conducted. recent advancements in sequencing technologies have led to an explosive growth in the analysis of infection-related genes of oomycete plant pathogens. the application of next generation sequencing techniques in non-model species promises to collect large amount of data necessary for functional studies rapidly and cost-effectively. in order to gain more insights into the molecular mechanisms of p. cactorum pathogenicity, in the present study we report on the illumina sequencing, de novo assembly, annotation and analysis of the p. cactorum transcriptome in important life cycle stages. a large number of genes and gene families were identified using different methods. we investigated the potential effector arsenal including rxlr effectors. the rxlr candidate effector dataset was searched for the conserved structure unit, wy-domain fold. transcript levels of potential effector genes were monitored during the developmental and host infection stages of p. cactorum. heterologous expression revealed that some effectors could elicit pcd. sequence polymorphisms and positively selected sites in rxlr effector candidates were also analyzed. in total, the work described herein provides a crucial foundation for further dissection of genes relevant to virulence in this broad-host-range phytopathogen.  sampling of p. cactorumlife cycle stages to gain a global overview of the p. cactorum transcriptome and gene activity during the important life stages, we prepared a mixed cdna sample using rna from different life stages: mycelia, sporangia, swimming zoospores, cysts and germinating cysts with germ tubes.figure life cycle stages of p. cactorum including cysts germinating under different conditions. five successive life cycle stages are shown: mycelia, sporangia, released sporangia and zoospores, cysts, - germinating cysts. in panel, the arrow indicates a zoospore being released from the sporangium. cysts were germinated on a cellophane membrane placed on the top of an n. benthamiana leaf, or directly on n. benthamiana leaves, or in water. in panel, the cysts were observed under a cryoscanning electron microscope. the other cell types were observed using an olympus system microscope bx photos were taken at, and min post-inoculation. scale bars:,, 10 μm;,,, 50 μm;, 100 μm. in this study, we found that p. cactorum can infect the model plant n. benthamiana at least through foliage and roots. the infection by p. cactorum reproducibly caused visible leaf cell death, observed at and 24 h post-inoculation using trypan blue staining. in order to investigate early infection events by p. cactorum but to avoid the involvement of plant tissue in the pathogen rna samples for later transcriptome sequencing, a cellophane membrane placed on the n. benthamiana leaf was used as a surface for the induction of cyst germination as previously described for magnaporthe grisea. the cysts germinating on both cellophane membranes and n. benthamiana leaves were morphologically similar, in contrast to cysts germinating in water.figure disease progressions in n. benthamiana caused by p. cactorum isolate leaves or roots were inoculated with a p. cactorum zoospore suspension. trypan blue-stained leaves at and 24 h post-inoculation. on each leaf, the left side of the main vein was mock-inoculated with water while the right side was inoculated with zoospores. plants were inoculated by pouring zoospore suspension around the roots. upper panel, disease development on whole plants 5 days post-inoculation. lower panel, root from plants in upper panel experiments were cut open to observe symptoms inside the roots. ctrl, roots from control plants mock-inoculated with water. to further test if the cysts germinating under this artificial condition display similar gene expression pattern to that of natural infection, rxlr candidate genes were selected for comparison with normalized read counts and real-time quantitative rt-pcr. during the transcriptome sequencing period, illumina rna-seq was also utilized to perform massively parallel sequencing of p. cactorum my, zo and gc in our lab. about million sequence reads for each library were mapped to the assembled reference transcriptome, and the transcript level of each gene in different libraries was calculated and normalized to the reads per kilobase of exon model per million mapped reads for comparison. at the same time, expression of these rxlr candidate genes’ mrna was measured using qrt-pcr under the artificial condition. a parallel qrt-pcr analysis was also performed with the cysts germinating directly on n. benthamiana leaves for comparison. in gc versus my, all these rxlr candidate genes showed the same expression pattern under artificial condition as during natural infection. this direct comparison faithfully demonstrated that genes differentially expressed in cysts germinating on cellophane show the same expression pattern as during natural infection, relative to the mycelium. these results convinced us that cellophane membrane was a valid model for the cyst germination. we did not harvest cysts germinating in water because in the absence of the leaf or membrane surface, cyst germination was significantly slower and non-uniform. hence, rna from the cysts germinating on cellophane membrane was extracted at 60 min post inoculation and mixed equally with the my, sp, zo and cy rna samples for illumina rna-seq. transcriptome sequencing and de novoassembly we used illumina platform to perform p. cactorum transcriptome sequencing. two biological replicates were generated and sequenced for mixed samples of five life cycle stages. each sequenced sample yielded 2 ×  or 2 × 83-bp independent reads from either end of a cdna fragment. after filtration of low-quality and adapter sequences, a total of, cleaned cdna reads was obtained and combined for analyses hereafter. the percentage of q bases for the clean reads were >94%. an average “g + c” content of above 56% was observed for the p. cactorum cdna sequences. an overview of the assembled contigs, scaffolds and unigenes is presented in table  initially, the reads were assembled into contigs. after paired-end joining and gap-filling, the contigs were further assembled into scaffolds totaling, bases. the final clustering of scaffolds produced unique genes. more than half of the unigenes were longer than 500 bp and unigenes were longer than kbp. the distribution of scaffolds and unigenes is shown in additional file for the randomly fragmented transcriptome, there was a positive relationship between the length of a given unigene and the number of reads assembled into it. to evaluate the completeness of the transcriptome assembly, we used the core eukaryotic genes mapping approach pipeline, with the cegma subset of the widely conserved core eukaryotic genes that are likely to have low frequencies of gene family expansion. using this tool, of the cegma genes mapped against the p. cactorum transcriptome assembly were identified. the high recovery of cegma genes suggests a high quality transcriptome assembly.table summary of transcriptome assembly for p. cactorum sequence annotation the unigenes were annotated by aligning them with the deposited ones in diverse databases including p. infestans, p. sojae, pythium, ncbi nr, ncbi nt, swiss-prot, trembl, interproscan databases. the best one was selected from the matches with an e-value of less than 10− in total, unigenes could be matched to the genes from at least one of public databases. the results demonstrated that of the unigenes, had significant amino acid sequence matches in the p. infestans database and retrieved significant hits in the p. sojae database while only could be mapped to pythium genes. in contrast, of unigenes could be matched to the sequences deposited in the ncbi nr database.table summary of functional annotation of the p. cactorum transcriptome to better define conserved genes in p. cactorum, protein sequences from the transcriptome assembly of this species and three completely sequenced phytophthora pathogens, p. infestans, p. sojae and p. ramorum, were clustered into gene families based on reciprocal pairwise sequence similarities. in total, protein sequences from these four phytophthora species were clustered into groups. of them, orthologous groups were shared by all four species. a total of orthologous groups for p. cactorum, for p. infestans, for p. sojae, and for p. ramorum were generated. about 12% of p. cactorum putative proteins did not have orthologues in the other three phytophthora species, of which protein sequences were clustered into paralogous groups within the species. the remaining predicted proteins are considered singletons because they do not cluster at all in this analysis.figure four-way-venn-diagram of the distribution of unique and shared gene families among phytophthora species. homologous proteins in p. cactorum, p. infestans, p. sojae and p. ramorum were clustered into gene families using tribemcl. numbers in individual sections indicate number of gene families. overlapping regions denote groups with at least two proteins of all species that are part of the intersection. the first number under the organism name gives the total number of clusters that the respective organism contributes to; the second number describes the total number of each organism’s gene models/sequences that go into clusters. the difference of the second number to an organism’s total input proteins gives the number of singletons. by march there had been p. cactorum nucleotide sequences including expressed sequence tags deposited in the ncbi nt database. in order to characterize these sequences, we queried them against our transcriptome assembly. blastn comparisons revealed that of nucleotide sequences excluding ests were mapped to p. cactorum unigenes. of mapped sequences, were matched to the same unigene u. in contrast, of ests were mapped to unigenes. out of them, some ests may be derived from the same gene and redundant ests were mapped to unigenes. six unknown ests turned out to be clustered into ribosomal rna unigene u. go annotation in terms of “biological process”, “molecular function” and “cellular component” was conducted for the annotated p. cactorum unigenes and, for comparison, gene models of p. infestans. based on sequence similarity, p. cactorum unigenes could be categorized into functional groups. as shown in additional file the distribution pattern of p. cactorum sequences in different go categories was quite similar to that of p. infestans genes. the unigenes were assigned with one or more go terms. for “cellular component”, “cell & cell part”, “organelle” and “membrane” terms are dominant. for “molecular function”, genes related to “binding” and “catalytic activity” are highly represented. regarding “biological process”, “metabolic process” and “cellular process” are the highly represented go terms. we found a few genes from terms of “antioxidant activity”, “signaling”, and “localization”. besides go analysis, the unigenes were subjected to a search against the cog database for functional prediction and classification. in total, of the sequences retrieving a hit from the ncbi nr database could be assigned to cog classifications. cog-annotated putative proteins were functionally classified into protein families involved in cellular structure, biochemical metabolism, molecular processing, signal transduction and so on. the cluster for “general function prediction” represents the largest unknown group, followed by “replication, recombination and repair”, “transcription” and “signal transduction mechanisms”. the following categories: “extracellular structures”, “nuclear structure” and “cell motility”, represent the smallest groups. in addition, unigenes were assigned to “defense mechanisms” and unigenes were assigned to “translation, ribosomal structure and biogenesis”. to identify the biochemical pathways that are active in p. cactorum, we mapped the annotated sequences to the reference canonical pathways in kegg. this is a complementary approach to categorize genes functions with the emphasis on biochemical pathways. the process predicted a total of pathways represented by a total of unigenes. a summary of the sequences involved in these pathways is included in additional file these predicted pathways represented the majority of biochemical pathways for compound biosynthesis, degradation, utilization, and assimilation, and pathways involved in the processes of detoxification and generation of precursor metabolites and energy. the pathways with most representation by unique sequences were “ribosome”, “chromosome” and “spliceosome”. searching and validation of the in silico-predicted p. cactorumeffector genes like other plant oomycete pathogens, p. cactorum presumably secretes a battery of virulence proteins to promote infection. the secretome for pathogenicity-related proteins was identified by comparison of p. cactorum unigenes against public databases. a total of effector genes with known or putative roles in virulence were identified. notable families of secreted proteins include nlps, cbels with carbohydrate binding domains, elicitins and elicitin-like proteins, pcf-like proteins, and protease inhibitors. our data indicate that p. cactorum exhibits the same kinds of pathogenicity-related effector proteins that have been found in other oomycetes.table gene families potentially implicated in plant pathogenesis in p. cactorum p. cactorum a p. infestans b p. sojae b p. ramorum b pythium ultimum b hyaloperonospora arabidopsidis b albugo laibachii b saprolegnia parasitica b athe number of “real” effector genes in our dataset is yet to be determined though obvious pseudogenes are omitted. bdata from other oomycete species are from haas et al. for p. infestans, tyler et al. for p. sojae and p. ramorum, lévesque et al. for pythium ultimum, baxter et al. for h. arabidopsidis, kemen et al. for a. laibachii, or jiang et al. for s. parasitica. counts of annotated pseudogenes are omitted. recently, many studies have shown a vast repertoire of apoplastic and cytoplasmic effector proteins including rxlr and crinkler families in oomycetes. the p. cactorum unigene dataset was searched for similarity to known oomycete effectors. this search identified potential rxlr effectors containing an rxlr or rxlr variant motif located downstream of the signal peptide. to find the potential rxlr effectors without similarity to hits in sequenced genomes of oomycete species, all putative orfs from the assembly were generated and translated to amino acid sequences from both strands. the predicted peptides were analyzed using the rxlr effector identification method. this yielded the same rxlr effector dataset as blastx comparison did. another search for the amino acid motif rxlr was performed on these predicted peptides. hmmer searches employing models described previously yielded five additional potential rxlr effectors. signal peptide analysis was conducted using signalp and four candidates were excluded because of low signalp hmm probability. these led to the final set of unique potential rxlr effectors. phylogenetic analysis of the phytophthora genus has shown that p. cactorum lies with p. infestans and p. parasitica in clade. comparison of our rxlr dataset against p. parasitica inra- genome was also performed to double check the effector searching. we mapped a total of effector candidates to p. parasitica rxlr genes, leaving that matched to hypothetical protein-encoding genes and one not mapped to p. parasitica. in contrast, mapped to p. infestans rxlr genes and only mapped to uncharacterized p. infestans genes or no hits. four candidates matched to uncharacterized genes or no hit of both genomes at the same time. those uncharacterized hit proteins were identified as not rxlr effectors because of absence of rxlr motif in the sequences. among the potential effectors, contained a conserved rxlr motif. ten contained a variant of rxlr motif that is similar to the pexel translocation motif of plasmodium falciparum effectors. the details of candidates containing rxlr and/or deer motif or variants are provided in table  these candidates include intact orfs and incomplete sequences. the remaining rxlr effector candidates are found not to contain either rxlr or deer motifs due to short sequences.table canonical and variant rxlr-deer motifs and the wy-domain found in p. cactorum transcriptome protein models a b b b c cd e cf f f e -value f f pcrxlr1 pcrxlr2 pcrxlr3 pcrxlr4 pcrxlr5 pcrxlr6 pcrxlr7 pcrxlr8 pcrxlr9 pcrxlr10 pcrxlr11 pcrxlr12 pcrxlr13 pcrxlr14 pcrxlr15 pcrxlr16 pcrxlr17 pcrxlr18 pcrxlr19 pcrxlr20 pcrxlr21 pcrxlr22 pcrxlr23 pcrxlr24 pcrxlr25 pcrxlr26 pcrxlr27 pcrxlr28 pcrxlr29 pcrxlr30 pcrxlr31 pcrxlr32 pcrxlr33 pcrxlr34 pcrxlr35 pcrxlr36 pcrxlr37 pcrxlr38 pcrxlr39 pcrxlr40 pcrxlr41 pcrxlr42 pcrxlr43 pcrxlr44 pcrxlr45 pcrxlr46 pcrxlr47 pcrxlr48 pcrxlr49 pcrxlr50 pcrxlr51 pcrxlr52 pcrxlr53 pcrxlr54 pcrxlr55 pcrxlr56 pcrxlr57 pcrxlr58 pcrxlr59 pcrxlr60 pcrxlr61 pcrxlr62 pcrxlr63 pcrxlr64 aeffectors were predicted from the unigene sequences. bhidden markov model probability, nn mean s score, and signal peptide length were predicted using signalp v. call sequence coordinates are given in amino acid residues. ddetermined as two or more d,e,n and/or q residues followed by a r or k residue: { }. eevidence codes for rxlr gene prediction: m, b, p, and r. fwy-domain was predicted using the hmm for the wy-domain as described in with the hmm score cut-off of. the “—” means that no wy-domain was predicted. we performed similar searches to identify crn effectors in p. cactorum. in total, potential crn proteins were identified. three other pccrns were not picked up due to too short orf or lacked the characteristic n-terminal ~50-amino-acid lflak motif. the majority of coding sequences lacked both 5′ and 3′ sequences. twenty lacked 5′ sequences while four lacked a stop codon and apparently lacked 3′ sequences. three sequences were complete, but only one were identified with a predicted signal peptide. pccrn was previously detected in a transcriptional study of p. cactorum where it was found up-regulated during cyst germination and host infection. the number of “real” effector genes in our dataset is yet to be determined although obvious pseudogenes were omitted. however, some of the predicted effector genes were validated in this study. twenty-three effector sequences including rxlrs, nlps and one pcf-like were selected to check the accuracy of the computational analysis. they were tested by pcr amplification using p. cactorum genomic dna. a total of amplifications produced a unique band of the expected size. one sequence, pcrxlr produced two amplified bands, but the brighter one exhibited the expected size, suggesting the lower band resulted from non-specific amplification. three primer pairs towards the rxlr candidate genes did not produce expected bands. these genes were then full-length cloned and sequenced, yielding the same sequences as transcriptome assembly data. due to the approximately 87% correct predictions, the in silico analysis in this study was considered as providing reliable results.figure pcr amplification of a subset of the predicted p. cactorum unigenes. pcr amplification was performed on p. cactorum isolate genomic dna with primer pairs, and the amplicons were electrophoresed on an agarose gel. lane marker, dl- dna marker ladder. gene names and categories are listed on the top of the gel picture. the star indicates a non-specific amplification band for pcrxlr lane water, water control with pcnlp primers. the wy-domain is conserved in about half of p. cactorumrxlr effectors recently, it was identified by structural biology that wy-domain is a structure unit conserved in many phytophthora and h. arabidopsidis rxlr effectors. we searched p. cactorum rxlr effector candidates using the hmm for the wy-domain as described previously. using the score cut-off of, we revealed that of of p. cactorum rxlr effector candidates contain wy-domain-like sequences. wy-domains can also be found in tandem repeats, and we identified candidate effectors with more than one wy-domain. two candidate effectors, pcrxlr and pcrxlr contain wy-domains. therefore, many p. cactorum rxlr effector candidates comprise either single or tandem arrays of wy-domains. the expression of most effector genes is induced during p. cactoruminfection our study showed that p. cactorum can infect the model plant n. benthamiana. no evident macroscopic symptoms were observed in the root until hpi. the disease progressed quickly in the plants, and by d post-inoculation all inoculated plants were wilting or dead. to investigate the effector gene expression pattern changes during host infection, the infected n. benthamiana roots were harvested at infection time-points. this assured that the whole infection period of p. cactorum on n. benthamiana could be investigated. cdna samples from four life stages were also obtained as described previously but with one modification in which here gc was induced directly on n. benthamiana leaves. hence gc sample used here is a mixture of microbe and plant materials. expression experiments were performed for effector genes including rxlrs, one elicitin and one nlp. those representative rxlr unigenes were chosen based on a consideration of the signal peptide confidence, the intact orf and rxlr-deer presence. the transcript levels in the rna samples were assessed using semi-quantitative rt-pcr. eleven of the designed rxlr primer pairs produced unspecific or no fragments for the infection samples, and were not included for further analyses. figure  shows the expression patterns obtained for the remaining rxlr and other genes. all these rxlr genes were up-regulated during the host invasion, relative to my that can be regarded as the vegetative growth stage. in particular, all but one rxlr gene were highly expressed during the earliest infection stages. of these early expressed genes, also showed induced expression at gc which is an essential pre-infection stage of p. cactorum. the differential expression of rxlr candidate genes in comparison between gc and my was further validated by qrt-pcr and rpkm analyses. two rxlr genes were previously detected in a transcriptional study of p. cactorum where they were expressed during strawberry infection and cyst germination. that result is consistent with the result obtained in this study.figure expression patterns of 19 p. cactorum effector genes during life cycle and infection stages. lanes: my, plate-grown mycelia; sp, sporangia; zo, zoospores; gc, germinating cysts directly on n. benthamiana; - n. benthamiana roots at, 96 hours post-inoculation; pl, mock-inoculated plants; w, control leaves inoculated with distilled water. gene names and categories are listed on the left and the amplicon sizes are listed on the right. all sizes were as expected, and the experiment was performed with at least three biological replicates. in addition, the expression of two other effector genes was examined. the elicitin gene pcell showed stronger expression both at my and during late infection of n. benthamiana versus at hpi. the nlp gene pcnlp however, showed expression only at zo and hpi. p. cactorumeffectors elicit plant cell death and are conserved across oomycete species expression studies indicated that we had identified infection-related effector genes in p. cactorum. to begin an investigation of the activity of those potential effectors in planta, we conducted heterologous expression assays in n. benthamiana using infiltration of a. tumefaciens cells with the vector pgr. these methods have been proved valuable in the characterization and determination of the function of a variety of effector genes. all of the aforementioned effectror genes were cloned into the transient expression vector pgr a known elicitor of cell death, the p. infestans pamp inf, was used as positive control. a. tumefaciens strains harboring gfp served as negative control. the elicitin pcell, nlp effector pcnlp and four rxlr effectors triggered leaf cell death in n. benthamiana as vigorously as inf. expression of pcrxlr pcell and pcnlp resulted in leaf chlorosis throughout the entire infiltration zone by dpi, followed by initiation of cell death at dpi. by dpi, the zone of infiltration was completely dehydrated. the cell death symptoms triggered by pcrxlr and pcrxlr developed about d after infiltration whereas the symptoms caused by pcrxlr developed about d after infiltration. in contrast, only mosaic symptoms due to pvx were observed following infiltration with the remaining rxlr effector genes or with gfp under the same conditions.figure transient assays of phytophthora rxlr, nlp and elicitin effectors in n. benthamiana. the p. cactorum elicitin pcell, nlp pcnlp, four rxlrs, p. capsici rxlr pcaprxlr and the alleles of pcrxlr and pcrxlr triggered plant cell death. the plant leaves were infiltrated with a. tumefaciens cells to express each of these effectors. agro-infiltration sites in each n. benthamiana leaf expressing inf were the positive controls. the other sites in leaves expressing gfp only, or infiltrated by 10 mm mgcl buffer only served as negative controls. pcaprxlr is the ortholog gene of pcrxlr in p. capsici. the alleles of pcrxlr in the isolates ph-1-14- and ph-1-8- are labeled as pcrxlr and pcrxlr, respectively while the allele of pcrxlr in pc as pcrxlr. photographs were taken or d after infiltration. the circles indicated the agroinfiltration areas. we performed multiple sequence alignment of these elicitors of cell death, i.e. four rxlr effectors, pcell and pcnlp to determine their conservation within oomycetes. based on the conserved features and domain organization of oomycete effectors, the presence of a canonical rxlr motif and an eer motif was identified at n-termini of p. cactorum rxlr effectors and their orthologs in other phytophthora species. blastx results showed that p. cactorum pcrxlr pcrxlr pcrxlr and pcrxlr have 34%, 73%, 32% and 41% identity to p. infestans rxlr homologues, respectively. in contrast, these four effectors displayed higher sequence identity to p. parasitica homologues. however, the p. parasitica homologues of pcrxlr and pcrxlr were annotated as hypothetical proteins in the inra- reference genome although the canonical rxlr-eer motif is present. except pcrxlr the other three rxlr effectors have homologues in p. capsici and p. sojae. noticeably, pcrxlr and pcrxlr showed high sequence similarity to p. sojae effectors avh and avh respectively, which have been identified to trigger cell death in n. benthamiana and soybean. as reported previously, the rxlr motif of pcrxlr pcrxlr pcrxlr and their orthologs in other phytophthora species is confined to position 30– amino acids. however, analysis methods employing this constraint most likely eliminate genuine effectors. in this study, the canonical rxlr motif is located between residues and in the amino acid sequences of pcrxlr and its orthologs. data mining revealed that the coding sequence of its p. capsici ortholog, hereafter referred to as p. capsici rxlr protein, is embedded in a cdna clone cbot37-c but not annotated in the p. capsici reference genome. we cloned pcaprxlr into pgr and performed transient assay as described previously. intriguingly, pcaprxlr also triggered cell death in n. benthamiana leaves about d after infiltration. the p. capsici orthologs of pcrxlr and pcrxlr did not induce pcd in n. benthamiana. the above results together suggest that these rxlr effector proteins from different phytophthora pathogen species trigger similar responses in plant families. one nlp effector of interest, pcnlp showed of amino acids conserved across four different phytophthora species and an additional oomycete, pythium ultimum, whereas were shared with p. parasitica, corresponding to 85% identity. the data showed that all aligned nlps contained two conserved cysteine residues which defined these proteins as type i nlp. we also noted that the central conserved ghrhdwe heptapeptide motif is present in all nlps. the elicitin pcell shared amino acids with four phytophthora species and py. ultimum, whereas of amino acids were conserved between p. cactorum and p. infestans. polymorphic sites in p. cactorumrxlr effector sequences the four rxlr genes that triggered pcd were examined in a collection of seven p. cactorum isolates, to determine whether sequence polymorphisms may exist in these genes. effector genes may exhibit a high level of polymorphism if they are subject to diversifying selection as a result of pressure from plant resistance genes. two of the four rxlr genes were found to exhibit non-synonymous polymorphisms that altered the amino acid sequences they encoded. variations in amino acid residues were identified in pcrxlr among the sequenced isolates. these residues were located at the c-terminus of the sequence. the same fragment was not amplified from the isolate pc by pcr, suggesting that the gene may be deleted or substantially altered in this isolate. noticeably, the effector sequence was identical among the three european strawberry isolates, and in the two persian walnut isolates. pcrxlr could be amplified from all strains but fin but showed only one polymorphic site at the n-terminus among those six isolates. in contrast, pcrxlr and pcrxlr showed no amino acid polymorphisms among the tested isolates. the ability of the different effector alleles to trigger pcd was tested. the alleles of pcrxlr amplified from the isolates ph-1-8- and ph-1-14- induced the same pcd as the one from the isolate did. similarly, pcrxlr allele in pc also produced the same symptom on plants as pcrxlr of the isolate did. the results indicated that the polymorphic sites in these rxlr effectors do not affect their ability to trigger plant cell death.figure sequence polymorphisms of rxlr effector alleles among p. cactorum isolates. the protein sequences of pcrxlr in six isolates were compared. the allele was not obtained from the isolate pc the protein sequences of pcrxlr in six isolates were compared. the allele was not obtained from the isolate fin the putative rxlr and eer motifs of both effectors were denoted by red boxes. their predicted signal peptide sequences were underlined. the polymorphic sites were marked by blue asterisks for the visualization. we further examined the strength of natural selection acting upon the effectors by estimating the ratio of the non-synonymous substitution rate to the synonymous substitution rate. we defined the boundary of the n and c termini of protein sequences as the 70th or 116th amino acid. the yn tool in the paml v software package was used to calculate the ratio of dn to ds. three representative sequences made up three pairwise comparisons for the test of pcrxlr there is no polymorphism in the n-terminus of pcrxlr but in the c-terminus. however, no significant evidence of selection pressure in the c-terminus of pcrxlr was identified though the pairs 10300/ph-1-8- ph-1-8-03/ph-1-14- displayed dn > ds and the pair 10300/ph-1-14- showed dn < ds. single polymorphism site was found in the n-terminus among alleles of pcrxlr although dn > ds, it may not be significant. consistent with these results, model comparisons using the computer program codeml did not reveal any positively selected sites for either of the rxlr effectors.table d n /d s analysis of pairwise comparisons of the rxlr alleles b b n s n /d s a p n s n /d s a p n s n /d s a p pcrxlr6 pcrxlr7 athe ‘∞’ in column dn/ds refers to pairwise comparisons in which dn > and ds = and ‘–’ refers to pairwise comparisons in which dn = and ds = bthe n-terminus refers to the sequence from the 1st to the end of deer, and the c-terminus refers to the sequence from the immediate residue after deer to the end, except the stop codon. discussion despite the worldwide economic impact of diseases caused by p. cactorum, little is known about the molecular basis of the pathogenicity of this species. the availability of the genome sequences from other pathogens and their host plants has greatly advanced our understanding of pathogen virulence and host resistance. davis and yu and shulaev et al. presented the first genetic map and a genome sequence, respectively, for woodland strawberry, which is one important host of p. cactorum. in contrast, a lag still exists in developing these resources for the hemibiotrophic pathogen p. cactorum. chen et al. provided a first overview of genes that may be involved in p. cactorum pathogenicity. however, the sequence data obtained in that study were limited. additionally, due to the short sequences and lack of a reference genome, cloning strategies such as rapid-amplification of cdna end had to be employed to get the full length genes for ests of interest. the sequence data created in this study therefore represent a substantial expansion of the transcriptome resources available for p. cactorum and a glimpse into its pathogenicity mechanisms. due to the common problem that contamination of plant material in infection-stage samples often makes it difficult to study the plant-microbe interactions, examining phytophthora life cycle stages such as germinating cysts may be a useful alternative for investigating the transcriptional changes of infection-related phytophthora genes. previous studies have shown that molecules involved in establishment of infection and elicitation of plant defenses are extensively expressed during the life cycle stages. hence, in the present study the p. cactorum transcriptome sequences were generated by illumina sequencing from five successive life stages, and assembled by de novo short read. to prepare the important pre-infection stage, the germinating cysts, a cellophane membrane was placed on n. benthamiana leaf to mimic natural infection and induce germination of cysts while minimizing contamination of plant material. microscopic analysis first demonstrated that germinating cysts on cellophane morphologically resemble the ones germinating directly on n. benthamiana leaves, in contrast to the ones in water. second, the gene expression pattern in cysts germinating under both mimicry and natural conditions was investigated and compared. in gc versus my, the rxlr candidate genes under mimic condition showed the same expression pattern as during natural infection. the direct comparison provided further justification that our mimic experiments closely mirror the infection process occurred in fields. thus, cellophane membrane was a valid model for the cyst germination. the in vitro inoculation method could be applied to other similar plant-pathogen interaction studies. using the cegma tool, of the conserved single-copy eukaryotic genes mapped against the transcriptome assembly were identified in our study. similarly, % and 95% of cegma genes were reportedly detected in a. laibachii and h. arabidopsidis genome assemblies, respectively. the high recovery of 98% of cegma genes suggests a high quality p. cactorum transcriptome assembly in the present study. comparisons to sanger-derived sequences from p. cactorum, and pcr validation further showed that the transcript assemblies are robust. comparison of assembled gene models to gene catalogs of other oomycete species by blastx analysis and functional annotation indicate that the sequences represent an extensive catalog encompassing a large proportion of the genes expressed in p. cactorum. in the study, we used the data from tribemcl to globally examine the conservation of p. cactorum genes across phytophthora species. overall, gene families contained sequences from all four species, representing about 65% of the total number of gene families identified in this study. of that, gene families were specific for p. cactorum and p. infestans. in contrast, gene families were specific for p. cactorum and p. sojae, and for p. cactorum and p. ramorum. this fits well with the fact that p. cactorum lies more closely with p. infestans than with others in the phylogenetic tree. among those putative p. cactorum proteins that lack homologs in other species, were grouped into paralogous groups. the remaining were considered singletons, as they lack homologs within their own proteome or in the other taxa. this number is a bit larger than the counterpart numbers from each of other three species. this could be due to the sequencing depth and therefore assembly in this study. the assembly quality of these sequences could be improved in the future by even deeper sequencing and by genome sequencing. alternatively, these proteins could potentially represent p. cactorum-specific orphan genes. however, more research is required to dissect this. on the basis of gene annotations and pathway analyses, the p. cactorum unigenes are predominantly involved in cellular and metabolic processes, the binding and catalytic activities and cellular components. similar results were found in p. infestans. in addition, overall unique transcripts were predicted to be involved in kegg metabolic pathways, with two major pathways comprised of over unigenes. the predicted pathways together with the gene annotations will be useful for further investigations of gene function in the future. potential apoplastic and host-translocated effectors could be predicted from the assembled transcript sequences. in the present study, p. cactorum genes were identified encoding transporter or effector proteins with putative roles in virulence, with a repertoire similar to other oomycete plant pathogens. the p. cactorum transcripts obtained here represent the genes expressed during important life stages, and may therefore not represent all genes that are present in the genome. the p. cactorum effector data set could be expanded by including additional life cycle and infection stages of p. cactorum. sequencing of the genomes of phytophthora plant pathogens has revealed a variable number of rxlr effectors in different species. we identified potential rxlr effectors in the p. cactorum transcriptome, of which contained a conserved rxlr motif. additionally, potential effectors contained a variant of rxlr motif that is similar to the pexel translocation motif of pl. falciparum effectors. plasmodium pexel domains can functionally replace the rxlr-deer region of avr1b from p. sojae and p. infestans avr3a. furthermore, mutation of the fourth position of the p. sojae rxlr motif did not affect translocation. of the p. cactorum rxlr effector candidates, sequences appeared to be complete. we examined representative effectors using both rt-pcr and pcr cloning. seventeen full length sequences were cloned and confirmed to be rxlr genes. the other sequences were not further analyzed due to unspecific or no bands amplified from infection-stage samples in rt-pcr assays. among these sequences, only one can not produce any bands from all the test samples. more specific primers towards these sequences should be designed to dissect this. and, more work is needed to determine the number of functional or “real” genes in this dataset though obvious pseudogenes were excluded. two rxlr genes out of rxlr gene candidates reported in a previous study were re-detected in this study. the others mostly discovered by effector-specific differential display technique have not been re-detected in this study. however, this is not surprising because effector genes could be differentially expressed at different stages in response to different conditions. the rxlr effector candidates were detected by rna-seq during the pathogen developmental stages in the present study while most of potential rxlr effectors were identified during the strawberry infection by esdd in the previous study. some effector genes could be stage-specific and therefore not detected in both cases. the c-terminal regions of oomycete rxlr effectors carry the biochemical effector activity and about half of these proteins share a conserved wy-domain. this wy-domain fold may be an adaptive structural unit that can support effector diversification to gain new functions and/or evade plant host immunity. using hmm–based sequence searches, wy-domain had also been detected in our p. cactorum rxlr effector candidates, with out of containing this fold. this strengthens that the wy-domain unit could be critical for the success of oomycete plant pathogens. however, the reason why the wy-domain is preserved and its contribution to the functions of the effectors are still unknown. future studies are required to help define the roles of the wy-domain fold in the virulence mechanisms of these pathogens. we examined the transcript levels of those representative effector genes by hemi-quantitative rt-pcr and observed high expression during the host infection for of them. nearly half of the rxlr genes were differentially expressed during the life cycle and early infection stages of the p. cactorum. the expression patterns of the rxlr effector genes are consistent with their expected roles in assisting the colonization of plant tissues by manipulation of host defense responses. to explore their predicted roles further, we tested if the rxlr effectors could cause phenotypic reactions when expressed in host plants and found that four effectors could elicit plant cell death. effectors from other oomycete plant pathogens, including avh avh avh from p. sojae, pcaprxlr from p. capsici, pscrxlr and pcqne from pseudoperonospora cubensis and members of the crn family from p. infestans and p. sojae, have also been shown to elicit similar phenotypes when transiently expressed in n. benthamiana. the counter-intuitive ability of oomycete effectors that are expressed during biotrophy to trigger cell death has been inferred to reflect the ability of the effectors to manipulate the plant defense responses and/or the ability of the plant defense machinery to initiate effector-triggered immunity. wang et al. showed that a minority of p. sojae rxlr effectors triggered cell death in the host, soybean, or in n. benthamiana, and that a majority of tested p. sojae effectors could suppress the cell death triggered by those effectors. thus during natural infection it is likely that such effectors do not actually induce cell death, but instead manipulate the plant immune system in other ways that promote infection. previous studies have shown how the primary sequences of many rxlr effectors have been shaped by positive selection, and how the genes themselves undergo accelerated birth and death evolution. two of the cell death-inducing effectors, pcrxlr and pcrxlr exhibited polymorphisms but no significant evidence of natural selection among seven p. cactorum isolates. the polymorphisms were localized basically to the c-terminal effector domains. generally the amino acid sequences of each of both rxlr effectors were identical within isolates from the same host while different among isolates from different hosts. these host-related patterns may have been molded by host preferences, geographic isolation and/or degree of specialization, since the rxlr secretome is at the front line in the evolution of the host-pathogen interaction. the polymorphic nature of these p. cactorum rxlr effectors reinforces that these genes may play an important role in the microbe-plant interaction. however, no residues under positive selection in these two rxlr effectors were identified. this is possibly because only seven p. cactorum isolates were investigated for sequence polymorphism. the alleles of pcrxlr and pcrxlr retain pcd-triggering activity although their sequences showed polymorphism. the p. cactorum pcd-inducing rxlr effectors are highly conserved across several phytophthora species. ectopic expression data showed that the p. capsici ortholog of pcrxlr and p. sojae orthologs of pcrxlr and pcrxlr also triggered pcd in n. benthamiana, emphasizing their importance. these pcd-inducing effectors maybe represent few rxlr effectors conserved across phytophthora species. whether such rxlr ortholog pair targets a common plant component needs further investigations. in this study, the genes for the elicitin pcell and the nlp pcnlp were also validated, cloned and transiently expressed in plants. both genes showed distinct expression during the developmental and infection stages. ectopic expression in n. benthamiana displayed that both genes can trigger plant cell death. recent studies on other phytophthora species revealed similar results. based on multiple sequence alignment, we found that these effectors are highly conserved across oomycete species. together, these results suggested their important roles in the pathogenicity of p. cactorum. future work should determine the number of functional or “real” genes encoding these effectors in p. cactorum and elucidate their functional roles in the pathogen’s virulence.  as a root pathogen, and because of its broad host range, p. cactorum is an attractive model for understanding phytophthora infection. sequencing of the p. cactorum transcriptome has provided a wealth of information about the proteins expressed during the important life stages, especially the rxlr effectors. this is the first publication using next generation sequencing technology for p. cactorum without genome information. we have demonstrated the reliability of the transcriptome assembly by cegma, pcr validation and rt-pcr analyses. a large number of genes including rxlr effector candidates and gene families of p. cactorum were identified. about half of the rxlr effector candidates are predicted to have a similar overall conformation, termed the wy-domain fold. additionally, we have demonstrated the identification and characterization of pcd-triggering rxlr, nlp and elicitin effectors. more studies will however be required to determine the roles of infection-related effector genes in the pathogenesis. the results will ultimately help to elucidate the mechanisms underlying the pathogenicity of p. cactorum and to design effective control strategies for the diseases caused by this broad host range pathogen. 
a subset of the effector genes were checked and validated by pcr amplification. single nucleotide polymorphisms for rxlr effectors were detected in a collection of p. cactorum isolates from different countries and hosts. transcriptional experiments indicated that effector genes were differentially expressed during the life cycle and host infection stages of p. cactorum. in the absence of genome sequence, transcriptome data is important for infection-related gene discovery in p. cactorum, as demonstrated here for the effector genes. we first demonstrated an in vitro inoculation method that can be used to mimic natural cyst germination on host plants. to gain insight into its repertoire of effectors, the p. cactorum transcriptome was investigated using illumina rna-seq. about half of the rxlr effector candidates were predicted to share a conserved structure unit, termed the wy-domain fold. by comparisons with public databases, % of the unique genes were annotated, including mapped to the gene models of the annotated relative phytophthora infestans. over million cdna reads were obtained for five life cycle stages and de novo assembled into unique genes. these effectors are highly conserved across oomycete species. using tribemcl, gene families conserved across p. cactorum and other three completely sequenced phytophthora pathogen species were determined. in silico analyses revealed that p. cactorum effector homologues including rxlr effector candidates matched known or putative virulence genes in other oomycetes. this study demonstrates the comprehensive sequencing, de novo assembly, and analyses of the transcriptome of p. cactorum life cycle stages. the first look at the transcriptome and effector arsenal of p. cactorum provides valuable data to elucidate the pathogenicity basis of this broad-host-range pathogen. ectopic expression in nicotiana benthamiana revealed that rxlr, elicitin and nlp effectors can trigger plant cell death.
 phytophthora cactorum, a hemibiotrophic oomycete pathogen, can cause destructive diseases on numerous crops worldwide, leading to essential economic losses every year. however, little has been known about its molecular pathogenicity mechanisms. to gain insight into its repertoire of effectors, the p. cactorum transcriptome was investigated using illumina rna-seq.  we first demonstrated an in vitro inoculation method that can be used to mimic natural cyst germination on host plants. over million cdna reads were obtained for five life cycle stages and de novo assembled into unique genes. by comparisons with public databases, % of the unique genes were annotated, including mapped to the gene models of the annotated relative phytophthora infestans. using tribemcl, gene families conserved across p. cactorum and other three completely sequenced phytophthora pathogen species were determined. in silico analyses revealed that p. cactorum effector homologues including rxlr effector candidates matched known or putative virulence genes in other oomycetes. about half of the rxlr effector candidates were predicted to share a conserved structure unit, termed the wy-domain fold. a subset of the effector genes were checked and validated by pcr amplification. transcriptional experiments indicated that effector genes were differentially expressed during the life cycle and host infection stages of p. cactorum. ectopic expression in nicotiana benthamiana revealed that rxlr, elicitin and nlp effectors can trigger plant cell death. these effectors are highly conserved across oomycete species. single nucleotide polymorphisms for rxlr effectors were detected in a collection of p. cactorum isolates from different countries and hosts.  this study demonstrates the comprehensive sequencing, de novo assembly, and analyses of the transcriptome of p. cactorum life cycle stages. in the absence of genome sequence, transcriptome data is important for infection-related gene discovery in p. cactorum, as demonstrated here for the effector genes. the first look at the transcriptome and effector arsenal of p. cactorum provides valuable data to elucidate the pathogenicity basis of this broad-host-range pathogen. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords phytophthora cactorumtranscriptometribemcleffectorwy-domaintranscriptional profiletransient expressionplant cell deathpolymorphismissue-copyright-statement© the author 2014 