BACKGROUND
plant-pathogen interactions are a fine interplay between prospective host and pathogen, involving the exchange of molecular signals that determine the outcome of the interaction.
the plant endeavours to detect invaders by deploying, as a first line of defence, pattern recognition receptors that recognise pathogen-associated molecular patterns  shared by a wide range of non-specialised microbes, with a resulting induction of a low-level defence response termed pamp-triggered immunity .
in response, pathogens evolve effectors that enhance a pathogen’s ability to cause disease, often by blocking or suppressing pti .
plants in turn evolve resistance  proteins that recognise a subset of these effectors either directly or by their actions on plant host targets.
thus the respective effector and r protein complements can contribute to the determination of host-range for any given pathogen.
the genus venturia belongs to the order venturiales, which is assigned to the class dothideomycetes  <cit> .
the dothideomycetes include many highly destructive plant pathogens, the venturiales being no exception.
venturia pathogens are relatively host-specific and infect selected members of the family rosaceae, perhaps the best known and most widely researched of which is v. inaequalis cooke  that causes the economically important disease apple scab  <cit> .
related species, for example v. pirina, v. nashicola, v. carpophila and v. cerasi, cause scab diseases of other rosaceae hosts: european pear, asian pear, peach and cherry respectively .
venturia species cause similar symptoms by adopting analogous biotrophic parasitic strategies .
during infection they penetrate the host cuticle directly and then develop stromata  in the cuticle and sub-cuticular space without penetrating host cells.
ultimately conidiophores and conidia differentiate from stromata and erupt through the cuticle resulting in the formation of dark, crusty, dry lesions  on leaves and fruit.
a fundamental question arises as to what determines the host range of these pathogens, especially given that they have similar modes of biotrophic parasitism and infect closely related host species.
a further level of complexity underlies pathogen/host species specificity in scab fungi, for example, certain isolates classified as v. inaequalis on the basis of morphological and molecular criteria, as well as their ability to mate, are unable to infect malus, but instead infect different rosaceous hosts, such as eriobotrya  and pyracantha   <cit> .
whether these isolates should be considered as separate species or formae speciales is still open to debate.
gladieux and associates  <cit>  suggested that there should be no sub-species delineation based on analysis of six nuclear loci.
these isolates are therefore very closely related phylogenetically, but have distinct host specificities.
host cultivar specificity has long been demonstrated in isolates of v. inaequalis that infect malus  <cit> ;  <dig> gene-for-gene pairings have been identified to date between races of v. inaequalis and cultivars of malus.
only two scab r gene loci in apple  have been fully characterised ; however, no effectors of v. inaequalis have been characterised to date.
work to identify the v. inaequalis effector repertoire that determines cultivar specificity, and most probably host specificity, has been impeded by the lack of a whole genome sequence for this species.
genome estimates for v. inaequalis range from 38 mb  <cit>  to 100 mb  <cit> .
the de novo transcriptome of a single isolate of v. inaequalis has been published  <cit> , with analysis of both in vitro and in planta transcripts.
in our study the whole genome sequences of four isolates of v. inaequalis  were sequenced using illumina sequencing technologies.
the v. inaequalis genomes were compared to a single v. pirina genome  <cit> .
given the extracellular in planta niche the venturia fungi occupy during parasitism it can be assumed that the majority of the factors that enable successful parasitism are first secreted into the sub-cuticular plant-pathogen interface.
therefore the secretomes of these isolates are of utmost relevance and so were compared to identify putative pathogencity or virulence factors that may have a role in host determination, with special emphasis on secreted enzymes and putative proteinaceous effectors.
RESULTS
whole genome assemblies and gene predictions
a total of 40 mb  to 61 mb  was assembled for malus-infecting v. inaequalis isolates.
the assembled genome size for the loquat scab pathogen  was  <dig> and 41 mb for the pear scab pathogen .
lack of sequencing of mate-pair libraries with long inserts contributed to the vi <dig> .
<dig>  assembly having the lowest n <dig> value  among the five isolates, while vp had the largest n <dig> .
a summary of the repeat content masked for each genome assembly is presented in table  <dig> and additional file  <dig> table  <dig> the whole genome assemblies of isolates of venturia inaequalis and v. pirina
venturia inaequalis
venturia pirina
malus × domestica
malus × domestica
malus × domestica
eriobotrya japonica
pyrus communis
number of predicted genesa
percentage completeness: partial b
percentage completeness: partial c
ahybrid with hints/ab initio gene predictions for vi1
bcegma analysis using both partial and full gene sequences  <cit>
cbusco analysis using both partial and full gene sequences  <cit>
dnote the recent race designation change reported for eu-b <dig>  <cit> , previously reported as race  by bus et al.
<cit>
gene models were predicted with augustus gene prediction software   <cit> .
the number of gene models in the v. inaequalis isolates ranged from  <dig>  to  <dig>  whereas there were  <dig>  identified in v. pirina.
to estimate the completeness of the assembled genomes, the assemblies were scanned for the  <dig> most highly conserved core eukaryotic genes using the core eukaryotic genes mapping approach   <cit> .
comparisons of partial or complete predicted translated proteins demonstrated that the v. inaequalis vi <dig> genome is the most complete, with only one or two core eukaryotic genes missing respectively.
the lowest representation of the  <dig> core eukaryotic genes in any genome was still high,  <dig> complete and another seven partial predicted proteins for v. inaequalis vil, indicating a reasonably complete whole genome sequence for all isolates.
in addition, a benchmarking universal single-copy orthologs  analysis was also conducted using busco_v <dig> b  <cit> .
this analysis indicated that the v. pirina genome is the most complete and of the v. inaequalis genomes vi <dig> and vil are the most complete when partial predicted proteins are considered, although on comparison of complete predicted proteins the vil genome is the most complete .
since vi <dig> was the most comprehensive v. inaequalis genome in terms of coverage when cegma and busco analyses were taken together, and is predicted to have the most complete set of avirulence effectors , this isolate was selected for preliminary expression  analyses.
the predicted secretomes of venturia inaequalis and v. pirina
to predict the secretome of each isolate, gene models were analysed with a pipeline of programmes .
the total number of genes encoding the predicted secretome for each genome is as follows:  <dig> in vi1;  <dig> in vi <dig> ;  <dig> in vi <dig> .
<dig> ;  <dig> in vil and  <dig> in vp.
the number of predicted proteins in the vi <dig> secretome is greater than that for the other genomes due to use of both ab initio  and hybrid  gene predictions in the analysis.
thakur et al.
<cit>  reported a lower predicted secretome number of  <dig>  however this was based on transcriptome data alone and, as such, direct comparison is problematic.
gene ontology analysis through an in-house annotation pipeline  was used to annotate predicted genes in each secretome; however, the majority of the predicted proteins had no gene ontology classification.
hence, for this reason, the carbohydrate active enzyme  analysis toolkit   <cit> , and similarity searches utilising blastp  <cit>  against the ncbi non-redundant  database  <cit>  were used in addition to gene ontology.
where conflicting designations occurred, cat, then gene ontology took precedence over similarity search designations.
the secretome repertoire, in terms of diversity and abundance of protein classes, was similar between all venturia isolates, with small, secreted, non-enzymatic proteins  dominating .
for comparative purposes the secretomes of three plant pathogenic fungi, parastagonospora nodorum , cladosporium fulvum and puccinia graminis f. sp tritici were also identified using the same pipeline of programmes using predicted proteins downloaded from the joint genome institute .
the size of the p. graminis f. sp tritici secretome  was significantly larger than that recorded for c. fulvum and p. nodorum .
the size of the vi <dig> secretome was similar to that of p. graminis f. sp.
tritici, whereas the remaining venturia secretomes were of a similar size to those of p. nodorum and c. fulvum.fig.
<dig> the secretomes of four isolates of v. inaequalis and one isolate of v. pirina.
annotations were based on gene ontology analysis including interrogation of ncbi refseq  <cit> , interpro  <cit> , uniref  <cit> , expasy uniprotkb/swiss-prot  <cit>  and expasy prosite  <cit> , cazyme identification using the cat server  <cit>  and blastp searches against the ncbi non-redundant database  <cit> .
small secreted proteins  include predicted proteins with similarity to known effectors and proteins with no known function, either with or without putative conserved motifs identified by pfamscan
orthomcl was conducted to assess similarity between the predicted venturia secretomes   <cit> .
a total of  <dig> isolate-specific singletons were identified  but are not included in the orthomcl clustering figure .
the venturia core secretome  consisted of  <dig> orthologous clusters, with cazymes  and non-enzymatic ssps  being the predominant classes .
the venturia pan secretome  totalled  <dig> representing  <dig> individual proteins , with many lineage-specific and host-specificity candidates identified, most of which were predicted to encode non-enzymatic ssps.
the majority of these ssps had no significant similarity to proteins in the public domain.
the v. inaequalis-unique  pan secretome totalled  <dig> representing  <dig> individual proteins , with a total of  <dig> representing  <dig> individual proteins  specific to apple-infecting isolates, with  <dig> of the clusters  found in all three isolates.
there were  <dig> singletons, but no unique clusters specific to the loquat-infecting isolate vil.
the v. pirina-specific secretome totalled  <dig> representing  <dig> individual proteins, with  <dig> unique protein clusters and  <dig> singletons.fig.
<dig> proteins in the secretomes of four isolates of venturia inaequalis and one of v. pirina.
similar proteins in each of the secretomes were identified by orthomcl- <dig> .
<dig>  <cit>  together with the markov clustering algorithm mcl-09- <dig>  <cit> .
similarity levels were calculated based on reciprocal blastp similarity searches between the protein sequences with an e value threshold of 1e- <dig>  figures within the boxes represent the number of proteins in each cluster, whereas figures outside the wheel are the number of clusters in each sector.
those proteins that are singletons within each secretome are not represented
two sets of rna-seq data, from libraries made with rna harvested from v. inaequalis vi <dig> grown in vitro  and vi1-infected apple seedling leaves , provided evidence of transcription for  <dig> % of the vi <dig> secretome.
the rna-seq data were also used to select a smaller subgroup of the vi <dig> secretome to analyse in greater detail .
these data were used as indicators of involvement in virulence or pathogenicity.
figure  <dig> shows representative stages of this material -inoculated apple leaves, as assessed by microscopic evaluation).
two approaches were used: first, overall vi <dig> gene expression levels were estimated by analysing fragments per kilobase of exon per million reads mapped  values.
this enabled a ranking of the genes in terms of overall expression during infection to be calculated at both two and seven dpi and the most highly expressed genes at either time point were selected to give the top 5% data set.
of the  <dig> genes identified,  <dig> were expressed at two and seven dpi,  <dig> at two only and  <dig> at seven dpi only.
in addition, a differential expression analysis detected  <dig> of the  <dig> genes that were up-regulated during growth in planta, compared with in vitro growth .
thirty-seven genes were in both the top 5% data set and up-regulated during infection gene sets, giving a total of  <dig> individual genes .fig.
<dig> microscopic evaluation of venturia inaequalis vi <dig> infection of susceptible apple leaves.
a two, and b seven days post inoculation, and c in vitro  showing stromatic growth habit
fig.
<dig> predicted proteins present in the vis set from the secretome of venturia inaequalis isolate vi <dig>  annotations were based on gene ontology analysis including interrogation of ncbi refseq  <cit> , interpro  <cit> , uniref  <cit> , expasy uniprotkb/swiss-prot  <cit>  and expasy prosite  <cit> , cazyme identification using the cat server  <cit>  and blastp searches against the ncbi non-redundant database  <cit> .
small secreted proteins  include predicted proteins with similarity to known effectors and proteins with no known function, either with or without putative conserved motifs identified by pfamscan .
the order of the categories in the legend is the same as that in the chart
carbohydrate-active enzymes  and putative non-cazyme cell wall degrading enzymes
the cazyme repertoire  was similar between all venturia secretomes, with  <dig> belonging to the core secretome .
of these, all isolates have between  <dig> and  <dig> proteins with multiple cazyme domains.
proteins were identified as being associated with carbohydrate-binding motif , carbohydrate esterase , glycoside hydrolase , polysaccharide lyase  and glycosyl transferase  domains.
of these, the classes with the greatest number of protein members across each genome were those with: cbm <dig>  cbm <dig> and cbm <dig> domains, with specificity for cell wall carbohydrates, and in terms of the latter two, plant cell wall specifically; ce <dig> domains with specificity for cutin; gh <dig> with specificity for plant cell wall pectin; gh <dig> and gh <dig> with specificity for β-glycans; and gh <dig> with specificity for pectin and hemicelluloses.
also identified, although not as numerous, were cazyme domain-containing proteins predicted to interact with fungal cell wall components including: ghs associated with β-glycans, α-glucan, chitin, and cbm domains specific for chitin  <cit> .
the majority of the cazymes were either present in orthomcl clusters that had a single enzyme from each isolate and were thus members of the core secretome set, or in clusters comprised of enzymes from only the four isolates of v. inaequalis, not v. pirina .
similar numbers of predicted proteins  were identified in the secretome sets, which were similar to enzymes that degrade non-carbohydrate cell-wall components, e.g.
ligninases .
peptidases and lipases
depending on the isolate,  <dig> to  <dig> predicted proteins were identified in the venturia secretomes with similarity to peptidases .
the majority of these were conserved in the core secretome or in isolates of v. inaequalis rather than v. pirina.
there were proteins predicted in the secretomes of only the v. inaequalis isolates that were similar to avr-pita, the avirulence protein from magnaporthe with zinc metalloprotease features that binds directly to its cognate r protein  <cit> , albeit with a relatively high similarity score of 8e- <dig> recorded when the pathogen host interactions database   <cit>  was interrogated.
in addition, a further similar protein was also encoded by the genome of v. pirina but was not predicted to be secreted.
there were also between eight and  <dig> proteins, depending on secretome, with similarity to proteases from candida and coccidioides following interrogation of protein sequences against phi-base.
predicted proteins with similarity to lipases were also identified in each secretome .
primary metabolism and putative proteins involved in redox reactions
proteins with a wide range of functionality in primary metabolism, e.g.
proteins involved in amino acid  or nucleotide metabolism; phosphatases and nitrilases, and unclassified enzymes with an alpha/beta hydrolase fold were identified.
depending on the isolate, there were between  <dig> and  <dig> proteins in this category in each secretome, most of which were in the core venturia secretome.
numerous  proteins with putative functions in redox reactions were identified in the venturia secretomes.
putative proteins associated with the cell wall, transport, signalling and secondary metabolism
as expected there were few proteins in the secretomes associated with the cell wall, transport, signalling and secondary metabolism, since these proteins would be eliminated in the secretome prediction pipeline .
the majority of these proteins were members of the core secretome set .
small secreted proteins
proteins classified as ssps  made up between  <dig> and 66% of each secretome .
for comparative purposes, three secretomes derived from publicly available genomes  were also screened for ssps using the same pipeline of programmes as those used to analyse the venturia secretomes.
ssps  made up 79% of the secretome of p. graminis f. sp.
tritici, and  <dig> and 28% of the secretomes of p. nodorum and c. fulvum respectively.
smaller ssps less than  <dig> aa in length predominated in the venturia and p. graminis f. sp.
tritici secretomes, especially those with a predicted signal peptide.
in contrast, ssps of less than  <dig> aa in length in the predicted ssp repertoires of c. fulvum and p. nodorum were dominated by those predicted to be secreted by a non-classical mechanism .
the majority of genes encoding ssps in vi <dig> were supported with evidence of transcription .
most ssps are novel to venturia  and most of these novel ssps were ≤ <dig> aa in length.
the vast majority of vis genes, 245/ <dig>  were predicted to encode ssps ,  <dig> of which were ≤ <dig> aa in length .
of these smaller ssps,  <dig> have two or more cysteines in the mature predicted protein.
larger secreted proteins  with no similarity to known proteins were also represented in each of the secretomes .
candidate effectors from the ssp set
the secretomes of venturia have several proteins with a putative role in binding hydrophobic surfaces including between  <dig> and  <dig> proteins with hydrophobic surface binding  domains  as identified by pfamscan , with similarity to the aspergillus oryzae hsba, hydrophobic surface binding, cell wall galactomannoprotein.
five putative hsba-like genes were identified amongst the vi <dig> vis gene set.
another gene up-regulated at seven dpi encodes a small predicted mature protein of  <dig> aa with four cysteines and shares predicted aa sequence identity  to bacterial chaplins, which are membrane-associated proteins known to have similar functions to hydrophobins  <cit> .
predicted proteins with similarity to bacterial chaplins are also present in vi <dig> , vil and vp, but not vi <dig> .
<dig> .
two or three proteins with similarity to fungal hydrophobins were identified in the secretomes of v. inaequalis isolates and four in the v. pirina secretome, on the basis of conserved cysteine patterns  <cit> .
none were included in the vis gene set.
the venturia secretomes included predicted ssps with similarity to known effectors from other fungi, including ecp <dig>  from c. fulvum  <cit> , avrlm <dig>  from leptosphaeria maculans  <cit>  and ave <dig>  from verticillium spp.
<cit> .
the venturia candidate effectors were most similar to these effectors in a reciprocal blast of the cladosporium, leptosphaeria and verticillium genomes.
in addition to the orthomcl clustering analysis, further similarity searches within genomes were employed with a more relaxed threshold of 1e- <dig>  to ascertain whether putative paralogues  were present.
single orthologues of ecp <dig> were identified in each of the venturia genomes analysed, corroborating the data of thakur et al.
<cit>  who reported an orthologue of ecp <dig> in the transcriptome of a single apple-specific isolate of v. inaequalis.
a search of the pfam database  <cit>  with the vi <dig> orthologue of ecp <dig> revealed three peptidoglycan-binding, lysin motif  domains.
at the aa level there is 42% identity between ecp <dig> and the v. inaequalis orthologues, whereas the v. pirina orthologue has slightly higher identity at 44%.
the vi <dig> and vil predicted proteins are identical, whereas that from vi <dig> .
<dig>  has one aa substitution compared with the vi1/vil sequence and that from vi <dig>  has four aa substitutions.
the vi <dig> ecp <dig> orthologue was expressed during growth in planta and in vitro, although expression was not sufficiently high to be in the vis set.
orthologues of avrlm <dig>  <cit>  and ave <dig> are present as large gene families in all of the venturia genomes examined.
the copy number of full length avrlm6-like genes varied among v. inaequalis isolates , while the v. pirina isolate had  <dig> copies.
further characterisation of the venturia avrlm6-like gene families is reported in shiller et al.
<cit> .
three avrlm6-like genes are present in the vis gene set.
ten full-length ave1-like genes and seven ave1-like pseudogenes, were identified in the whole genome sequence of v. inaequalis vi <dig>  with similar numbers in all other venturia genomes.
the v. inaequalis ave1-like full length, predicted proteins range in similarity from  <dig> to 57% identity to v. dahliae ave <dig>  all but one of the vi <dig> ave1-like proteins have transcript evidence, with expression at all growth stages, including in vitro , with some very highly expressed during both infection time points.
two ave1-like gene loci are represented in the vis gene set.
loci of all  <dig> of the previously identified v
.
i
naequalis
candidate effector  genes  <cit>  from vi <dig> were identified in the vi <dig> wgs.
however, the majority of the predicted gene models associated with these loci were longer than those identified using ests  <cit> .
one vice gene  did not correspond with any augustus gene prediction.
only six vice predicted proteins were included in the secretome of vi <dig> , five of which were in orthomcl clusters in the core secretome, with a single predicted protein from each isolate in each cluster.
the sixth predicted protein  belongs to a cluster with members from only vi <dig>  and vp .
genes encoding four of these proteins were in the vis set, including the vice  <dig> gene.
the remaining nine vice gene models, assigned by augustus, did not encode proteins that were predicted to be secreted.
inspection of these loci revealed that, in the majority of these cases, the n-terminus predicted by augustus was upstream of the translation start site predicted by bowen and associates  <cit> .
thus the gene predictions at these loci need further validation by sequencing full length cdnas.
most of the vice gene transcripts were also detected by thakur et al.
<cit> , in a single transcriptome of v. inaequalis; however, these authors did not confirm whether these transcripts encoded predicted secreted proteins.
two cellophane-induced  genes, cin <dig> and cin <dig>  that encode proteins with internal repeats, have been previously identified as being highly expressed during both in vitro growth in cellophane membranes  and infection of vi <dig> on apple   <cit> .
the rna-seq data supported the expression levels reported in the previous study for both of these genes.
there were cin <dig> orthologues in each of the isolates examined forming a single orthomcl cluster, whereas cin <dig> orthologues were only in v. inaequalis isolates, again forming a single orthomcl cluster.
cin <dig> was one of the most highly expressed genes in the vis gene set, with very high expression in vitro as well as at both infection time points.
two additional genes, which encode smaller cysteine-rich cin1-like proteins, cin1l <dig> and cin1l <dig>   <cit> , are also found in each of the isolates.
however, their sequence is more variable since a single cluster with representative proteins from each isolate was not observed.
the proteins similar to cin1l <dig> from each v. inaequalis isolate clustered together, but that from the v. pirina isolate was classified as a singleton.
there are proteins similar to cin1l <dig> in each isolate; however, those proteins encoded by the vi <dig>  and vi <dig> .
<dig>  genomes, although having a signal peptide, were classified as being targeted to the mitochondria by both protcomp and wolf psort, and the proteins from the remaining isolates formed a single cluster.
cin1l <dig> and cin1l <dig> are in the vis gene set.
of those proteins originally classified as ssps in the venturia secretomes four shared similarity to proteins in phi-base  <cit> : the hydrophobin mhp <dig> from magnaporthe oryzae  <cit> ; npp <dig> from hyaloperonospora arabidopsidis ; gas <dig> from magnaporthe oryzae  <cit> ; bec <dig> from blumeria graminis f. sp.
hordei  <cit> .
none of these proteins were members of the vis set, although all had evidence of transcription in vitro and in planta .
ssp families in the vis set
of the  <dig> vis ssps ≤ <dig> aa in length in the vi <dig> secretome,  <dig> appear to be single proteins following spectral clustering analysis  <cit> ,  <dig> belong to  <dig> different families, having between two and  <dig> members.
the families include predicted proteins without in planta up-regulated expression of their corresponding genes and putative proteins that were not predicted by the original gene prediction software .
the remaining  <dig> ssps were not retained in the dataset since, although initially classified as an ssp ≤ <dig> aa in length, they belong to families where the majority of members have a robust annotation to an enzyme based on gene ontology analysis and similarity searches, or are larger than  <dig> aa in length.
family  <dig>   <dig> and  <dig> are very closely related and as an indication of this these families share members .
a representative sequence logo for family seven is shown in additional file  <dig>  although sequence diverse, the families have a putative conserved structure based on conservation of cysteines.fig.
<dig> small secreted proteins  in the venturia inaequalis vi <dig> secretome encoded by single genes.
only ssps ≤ <dig> amino acids in length are included.
the number of similar proteins in the venturia and related dothideomycete proteomes are indicated by numbers: black indicates a gene predicted by augustus, white indicates a putative coding sequence identified by tblastn using the protein sequence as query, followed by manual curation.
percentage identity is represented by: red = 100%; orange = 90–99%; yellow = 70–89%; green = 50–69; blue = 30–49%
fig.
<dig> small secreted protein  families in four venturia inaequalis and one v. pirina secretomes.
only ssps ≤ <dig> amino acids in length are included.
red = proteins in the same family from vi1; yellow = protein families from vi <dig> ; green = families from vi <dig> .
<dig> ; blue = families from vil; purple = families from vp.
families f1-f <dig>  and f34-f <dig> are cysteine rich i.e.
two or more cysteines per protein; f29-f <dig> are those with one or no cysteines.
★ = families with predicted similar proteins in related dothideomycete genomes
thirty families of predicted ssps, and  <dig> single proteins, have similar proteins present in all of the venturia isolates analysed.
all, except five, of the single proteins  are also single proteins in all of the isolates studied.
seven families are unique to and conserved within the isolates of v. inaequalis, whereas there are  <dig> single proteins in vi <dig> with similar proteins present in all v. inaequalis isolates and absent from v. pirina.
there is one family, and two single proteins, restricted to isolates that only infect apple .
in addition, there are four single ssp proteins, and one family, that are putative candidates for the effector/s that contribute to the race profile of isolate vi <dig> .
<dig> , i.e.
present in vi <dig> and vi <dig> , but absent from vi <dig> .
<dig>  .
there is one family and three single proteins that are candidates for effector avrrvi <dig>  given their presence  and absence  in the venturia isolates.table  <dig> small secreted proteins  in the venturia infection secretome  set and related fungi
both families and single genes encoding small secreted proteins ≤ <dig> amino acids in length are included.
families were analysed by spectral clustering followed by manual curation and analysis of non-predicted putative genes
aticks in columns indicate presence of a similar protein or proteins in that secretome
to ascertain whether any of the  <dig> ssps of the vis set were conserved across the dothideomycetes, similarity searches  were made against selected dothideomycete predicted proteomes curated at the jgi   <cit> ; this curation includes clustering information pertaining to proteins from each organism.
only ssps predicted for the vi <dig> secretome, which were present in all the venturia isolates, had putative similar members in related dothideomycete predicted proteomes .fig.
<dig> similar small secreted proteins  in dothideomycete proteomes and the venturia inaequalis vi <dig> secretome.
only ssps ≤ <dig> amino acids in length are included.
shades of blue, purple and pink = pleosporales: cochliobolus sativus, c. heterostrophus c <dig>  c. heterostrophus c <dig>  c. lunatus, c. miyabeanus, c. victoriae, pyrenophora tritici-repentis, p. teres f. teres, leptosphaeria maculans, parastagonospora nodorum; shades of orange, yellow and red = capnodiales: septoria populicola , s. musiva , m. fijiensis, cladosporium fulvum , dothistroma septosporum, zymoseptoria tritici, baudoinia compniacensis; green = dothideales: aureobasidium pullulans var.
pullulans
putative host range determinants in the ssp set
in addition to those candidate effectors that are specific to either v. inaequalis, or those isolates able to infect apple described above, ssps ≤ <dig> aa in length specific to the loquat-infecting isolate of v. inaequalis or to v. pirina were identified by orthomcl analysis .
in addition to the  <dig> candidate effectors specific to vil, only four of which had any similarity to proteins in the ncbi nr database,  <dig> were found in both the vil and vp secretomes.
a further  <dig> candidate effectors were found to be specific to v. pirina.
gene density and proximity of genes to transposable elements
most genes in the vi <dig> genome have intergenic regions between 200 bp and 3 kb , with a mean intergenic distance of 1677 bp.
a subset of core eukaryotic genes  <cit>  had a slightly shorter mean intergenic distance of 1289 bp, whereas the ssp genes in the vis set , display a significantly  longer average intergenic distance at 2560 bp.
genes located on scaffold ends, hence lacking neighbouring genes, were excluded from this analysis.
this excluded 17/ <dig>  from the core eukaryotic gene set and 21/ <dig>  from the ssps in the vis set.
the greater proportion of ssp genes located on the ends of scaffolds suggests a bias for association with complex repeat regions that cause problems with sequencing and assembly, resulting in broken genomic scaffolds.fig.
<dig> flanking distance  between predicted genes of venturia inaequalis vi <dig>  intergenic distances for all predicted genes are represented in the underlying heatmap, with the number of genes in each bin shown as a colour-coded heat map  generated as in saunders et al.
<cit> .
genes were sorted into two-dimensional bins on the basis of the lengths of flanking intergenic distances to neighbouring genes at their 5′ and 3′ ends; overlying this are scatterplots of a  <dig> core eukaryotic genes  or b
venturia infection secretome  gene set, plus avrlm6- and ave1-like genes ; light pink = ssps with one or no cysteines ; blue = peptidases; dark green = cazymes; light green = putative cell wall-degrading enzymes ; white = cell wall associated and miscellaneous proteins > <dig> amino acids).
note that the axes are not linear.
genes at the scaffold end were excluded from this analysis
concordantly, ssp genes also appear to be more closely associated with transposable elements  or te-like features than core eukaryotic genes and all remaining genes in the vi <dig> genome.
the mean gene to te distance was 9024 bp for all genes predicted in the genome .
the core eukaryotic gene set had a significantly  greater mean distance to the nearest te of  <dig>  bp, whereas the ssps in the vis set had a mean distance to the nearest te  of only 5401 bp.
twenty-two ssps in the vis set were nested within predicted te-like features.
fourteen of the core eukaryotic genes and six of the vis set ssp genes were excluded from this analysis as they lacked a neighbouring te feature on the same scaffold.fig.
<dig> distance between predicted genes and transposable element -like features of venturia inaequalis vi <dig>  flanking distances  to te-like features for all predicted genes are represented in the underlying heat map, with the number of genes in each bin shown as a colour-coded heat map on orthogonal projection .
genes were sorted into two dimensional bins on the basis of the lengths of flanking distances.
a  <dig> core eukaryotic genes  are similar to all genes in that they are not closely associated with te-like features; whereas b
venturia infection secretome  gene set, plus avrlm6- and ave1-like genes ; light pink = ssps with one or no cysteines or less ; blue = enzymes ; dark green = cazymes; light green = putative cell wall-degrading enzymes ; white = cell wall associated and miscellaneous proteins > <dig> amino acids in length).
note that the axes are not linear.
genes at the scaffold end were excluded from this analysis
discussion
comparisons of whole genome assemblies and gene predictions
this paper reports the first analysis of whole genome sequences from multiple isolates of v. inaequalis   <cit> .
the sizes of all five venturia genomes are comparable to those of other dothideomycetes  <cit> ; however, these may be an underestimate since the majority of the sequencing was carried out on the illumina platform.
assembling repeat regions using short reads is notoriously difficult , and hinders genome assembly using de bruijn graph-based genome assemblers like velvet and allpaths-lg  <cit> .
in addition, there is an apparent wide range of genome size that can be largely attributed to additional repetitive content, with the assembled genic regions being similar in size for all five venturia isolates.
whilst variation in genome size between isolates from the same species is not unprecedented; for example, genome size in three isolates of the dothideomycete zymoseptoria tritici  ranged from  <dig> to 40 mb as estimated by pulsed field gel electrophoresis  <cit> , this apparent variation in the venturia genomes may be attributable to the different strategies, and software, used for sequencing.
the genomes  assembled using allpaths-lg software, have much larger genome sizes and higher percentages of repeats, than those assembled using velvet .
the major difference between the sequencing of these five isolates is that vi <dig>  and vil paired-end  libraries were specifically constructed with an insert size of 180 bp so that paired reads overlapped at the end , to produce longer super-reads.
for the other three isolates, the pe libraries were constructed with a typical insert size of 400 bp, and paired reads did not overlap and could not be assembled using allpaths-lg.
the super-reads generated by allpaths-lg result in repeats being better assembled  <cit> .
as a trial, in addition to allpaths-lg, the vi <dig>  and vil sequences were also assembled using velvet.
the percentage of repeats in the velvet scaffolds for each isolate was lower than that for the allpaths-lg assembly, but was still very high compared with the remaining three isolates, with  <dig>  and 25% for vi <dig>  and vil respectively.
additional investigation is required to determine whether the larger genome sizes and higher percentages of repeats in vi <dig>  and vil are a biological reality.
the number of predicted genes in the venturia genomes  is significantly less than the  <dig>  unique fungal genes reported for v. inaequalis by thakur et al.
<cit> .
just over half of the reported sequences had no significant similarity to sequences in other species.
the thakur et al.
<cit>  estimate was based on in vitro and in planta transcriptome data only, with multiple gene splice variants likely to account for the higher gene number.
our estimate of predicted gene number is comparable to related dothideomycete genomes which range from  <dig> in s. populicola  <cit>  to  <dig>  in c. fulvum  <cit> .
gene number variation could be due to difference in coverage of the various genomes; however, variation in gene number between isolates is also not unprecedented.
for example, xue and associates  <cit>  found that in the rice blast fungus m. oryzae, gene number varied, with hundreds of isolate-specific genes present in genomes of field isolates.
similar gene number variation was reported in isolates of c. heterostrophus  <cit> .
venturia pathogens: lifestyle and host determination
focussing on the secretome of the venturia pathogens has revealed repertoires of proteins that reflect aspects of their adopted mode of parasitism, with secretion of compounds to adhere to the waxy, water-proof cuticle on leaves and fruit and to enable direct penetration and colonisation of the cuticle and sub-cuticular space.
in addition, effectors that are predicted to effect evasion of recognition and suppression of host defence will be secreted to the extracellular plant-pathogen interface, including those that may be directed to be taken up by the host cells.
attachment and penetration
several venturia proteins were identified in the secretomes that may be involved in attachment and penetration of the cuticle.
a predicted protein present in all the dothideomycete genomes analysed has similarity to a m. oryzae fasciclin protein.
fasciclins have been implicated in cell adhesion in diverse organisms including both prokaryotes and eukaryotes .
the m. oryzae fasciclin has an important role in development and pathogenicity, involved in conidiation and conidial adhesion  <cit> .
a similar role for fasciclins in cell adhesion for all the venturia isolates analysed is easily envisaged.
hydrophobins are small, secreted, cysteine-rich, amphipathic proteins that are usually found on the cell walls of fungi  <cit> .
they provide a water-repellent coat and are well characterized for their role in morphogenesis and virulence in plant pathogenic fungi, promoting interactions with hydrophobic surfaces  <cit> .
it is thought that these proteins may also assist in avoiding detection by the host during infection  <cit> .
hydrophobins are usually highly variable in sequence; however, they have a conserved structure due to conserved cysteine residues.
venturia secretomes have fewer hydrophobin genes  than c. fulvum  but have a similar number to d. septosporum   <cit> .
the venturia secretomes also have a high number of genes  encoding putative hsba proteins.
hsba proteins are found in entomopathogenic fungi as well as pathogens of higher animals; with cuticle colonisation being a possible common link.
hsbas are not commonly reported to be encoded by plant pathogen genomes .
one sequence with similarity to an hsba was identified in the genome of the mesophyll apoplast-dwelling c. fulvum and the pine pathogen d. septosporum  <cit> .
hsbas are not hydrophobins, but they have been reported  <cit>  to have an analagous role in a. oryzae in binding to hydrophobic surfaces and recruiting the cutl <dig> polyesterase/cutinase to degrade the polyester substrate, poly .
takahashi and associates  <cit>  also reported that the a. oryzae hydrophobin, rola, also recruits cutl <dig>  to aid degradation of pbsa surfaces.
thus, a. oryzae appears to use several types of proteins to recruit lytic enzymes to the surface of hydrophobic solid materials and promote their degradation.
proteins similar to the a. oryzae cutl <dig> polyesterase/cutinase and hsba were well represented in the vis set.
extracellular cutinase has been implicated in penetration by v. inaequalis, as cutinase is produced by germinating conidia, and a cutinase inhibitor can prevent penetration  <cit> .
in addition, esterase-like activity has been reported during the germination of conidia and in appressoria  <cit> .
the presence of high numbers of ces in the venturia secretomes supports the experimental evidence of enzymatic penetration of the cuticle by v. inaequalis and the inference that v. pirina also may act similarly.
we propose that venturia, like a. oryzae, uses multiple proteins  to recruit cutinases/esterases to facilitate appressorial adhesion and direct penetration, as well as degradation and digestion of cuticle.
this hypothesis fits observations of the cuticular degradation that occurs during colonisation by venturia fungi potentially drawing nutrition from the cuticle or cuticle precursors  <cit> .
nutrition
exploitation of the cuticle may be insufficient to satisfy pathogen nutritional requirements throughout the infection cycle.
during biotrophic infection venturia remains in the cuticle and sub-cuticular space causing relatively little damage to the host.
most damage is due to breaching of the cuticle upon sporulation  <cit> .
at this time the epidermal cells underlying the stroma undergo a progressive depletion of plastids and cytoplasm, accompanied by increasing vacuolation, leading ultimately to cell death.
it has been proposed that this damage late in the infection cycle is caused by partial cell wall degradation  <cit> .
indeed, cellulase and pectinase activities have all been reported for v. inaequalis growing in vitro  <cit> .
however, it has been suggested that the timing of this host cell degradation precludes a significant role for these enzymes in nutrient acquisition  <cit> .
the predominant polysaccharide in apple fruit skin is pectin at 65%, compared with a level of 3% for cellulose  <cit> , with pear fruit also having higher levels of pectin than cellulose  <cit> .
venturia appears to have tailored its cazyme repertoire to suit the composition of the host cell wall with pectin-specific cazymes predominating; the two most numerous classes of cazymes found in the venturia secretomes, after those with cutinase activity, are gh <dig> and gh <dig> that have pectin as a substrate.
a single gh <dig> enzyme and two putative pls, that also have pectin as target substrate  <cit> , are up-regulated during infection and are present in the vis set.
significantly, this lytic activity may therefore not be limited to late in the infection cycle as previously thought, but contribute to nutrition via degrading the surface polysaccharides of the epidermal cells beneath stromata, this damage only becoming evident macroscopically late in infection.
in addition, those cazymes present in the secretomes of the venturia fungi may also benefit the fungus during saprobic growth and sexual fruiting body development during winter.
evasion of host defence
the putative proteases present in the secretomes of the venturia fungi may aid in the evasion of host defense since proteases have been implicated in plant defence avoidance in many plant-pathogen interactions  <cit> .
for example, fungal proteases can target plant chitinases, as in the interaction between fusarium oxysporum f. sp lycopersici and tomato where both a serine protease and a metalloprotease are required for the inactivation of a chitin-binding domain -containing chitinase thus contributing to virulence  <cit> .
as a possible alternative strategy to counteract host chitinases the venturia strains, in common with many other fungi, all have a homologue of the c. fulvum lysm domain effector, ecp <dig>  these are likely to either have a similar function to c. fulvum ecp <dig>  <cit>  in preventing recognition of chitin fragments capable of initiating a defence response, or to the lysm effectors from z. tritici that protect hyphae from the action of chitinases, thus preventing the release of chitin-derived pamps  <cit> .
in addition, there are single proteins in each secretome similar to the blumeria effector bec <dig>  <cit> .
the putative venturia bec <dig> orthologues all have the conserved etvic motif that is required for suppression of the hypersensitive response  in barley.
a similar function for these proteins during the pathogenicity of venturia is therefore easily envisaged.
although single proteins similar to the npp <dig> protein from h. arabidopsidis are present in each of the venturia secretomes  <cit>  the sequence of these from two apple-infecting isolates are truncated.
in the remaining secretomes  the npp1-similar proteins appear to belong to the type i nep-1-like protein  family, with the two conserved cysteines  <cit> .
all three proteins also have two additional c-terminal cysteines, however these are not characteristic of type  <dig> nlps  <cit> .
the majority of nlps induce necrosis in a wide-range of dicotyledonous plants, although whether this necrosis is a direct result of cytotoxicity via dispruption of the plasma membrane or initiation of a defence response remains open to debate  <cit> .
however, the requirement for a functional necrosis-inducing protein in the biotrophic venturia pathogens is unlikely.
indeed, each of the venturia proteins has mutations in the critical h residue in the loop region required for necrosis in the nlp from v. dahliae  <cit> , whilst the two v. inaequalis proteins also have a mutation in the heptapeptide domain  required for necrosis in nlps from diverse taxa  <cit> .
these mutations may therefore prevent unwanted pathogen-induced necrosis, but the retention of these nlp-like proteins by venturia may indicate a virulence function that remains to be elucidated.
venturia predicted proteins with similarity to the magnaporthe gas <dig> protein, required for appressorial penetration and lesion development, may play a similar role in apple and pear scab diseases to that observed in rice.
whether gas <dig> is translocated to the host cytoplasm with a role in defence suppression remains equivocal, however; weak fluorescence of fluorescent protein constructs in infectious hyphae in onion epidermal cells was observed  <cit> , as was translocation to rice cytoplasm, albeit under, presumably, the heterologous protomoter from the magnaporthe gene pwl <dig>  <cit> .
the venturia proteins may therefore be translocated to the host cell cytoplasm where they may have a role in re-directing host metabolism during scab disease, however this remains conjecture.
initiation of host resistance: an avirulence function
the comparative approach undertaken here has enabled preliminary identification of candidates for v. inaequalis avirulence effectors avrrvi <dig>  avrrvi <dig>  avrrvi <dig> and avrrvi <dig>  which have an avirulence function and are recognised by cognate r proteins.
all candidates have cysteine residues but no recognisable protein motifs, except a single candidate for avrrvi <dig>  avrrvi <dig> or avrrvi <dig> that is similar to a bacterial chaplin with a domain of unknown function, that may play a role in the venturia lifecycle similar to that of hydrophobins.
in addition to ssps that may be involved in race/cultivar specificity on apple within v. inaequalis, further ssps were identified that are specific to the loquat-specific isolate of v. inaequalis or to v. pirina.
the majority of these ssps were either similar to hypothetical proteins available in the public domain or had no similarity with known proteins.
however, of interest is a protein with ankyrin repeats that may be involved in the specificity of v. pirina, since it is dissimilar to proteins found in the other secretomes.
ankyrin repeat-containing proteins are involved in protein-protein interactions and are present in effectors from both prokaryotes and eukaryotes, and as such this protein may play a role in the interaction with european pear  <cit> .
in addition, a single protein specific to the vil isolate has a snoal-like domain.
snoal-like domains are found in proteins from diverse taxa, including filamentous fungal phytopathogens.
for example, the protein pep <dig> from nectria haematococca mpvi has such a domain and contributes to virulence on pea; complementing an isolate of n. haematococca lacking a supernumerary chromosome bearing pathogenicity genes with pep <dig> results in a small, but significant, increase in virulence  <cit> .
how these candidate effectors may be contributing to host range determination is unknown.
they may be acting as avirulence proteins during attempted infection of a nonhost, with a recognition event triggering resistance, and a concomitant lack of recognition in a compatible host.
indeed, since the hosts are closely related  <cit> , the contribution of r protein-triggered immunity towards nonhost resistance has been postulated to be greater than for host plants that are more distantly related  <cit> .
the variability of the closely related genomes of the venturiaceae with respect to ssps reinforces the utility of a comparative genomic approach.
in each of the v. inaequalis secretomes the predicted proteins with similarity to avr-pita <dig> from m. oryzae may play a similar role to that adopted by both avr-pita <dig>  and its paralogue avr-pita <dig>  in the rice blast interaction  <cit> .
although the virulence function of avr-pita <dig>  a putative zinc metalloprotease, has not been verified it is directly recognised by the cognate r gene product pi-ta.
an avirulence function for the similar proteins in the venturia pathogens remains to be elucidated.
effectors with unknown function
in addition to those proteins with similarity to known effectors with roles partially or fully confirmed, the venturia secretomes comprise a large repertoire of putative effector ssps with no obvious hints as to function from similarity searches.
the paradigm for the role of ssps is that they are lineage-specific effectors that contribute to the maintenance and evolution of parasitism and that obligate biotrophy is associated with expanded effector repertoires  <cit> .
it is thought that biotrophs require a more extensive and nuanced effector inventory than that required by necrotrophs, that rely more prominently on cell-wall degrading enzymes and toxins  <cit> .
although this précis of fungal pathogen lifestyles is highly simplistic, it seems to be reflected in the fewer number of ssps recorded for necrotrophic fungal phytopathogens.
for example parastagonospora nodorum has a suite of only  <dig> ssps  <cit> , and a recent analysis of the secretome of sclerotinia sclerotiorum revealed only  <dig> effector candidates, albeit using different criteria for selection  <cit> .
this contrasts with the numbers of ssps reported for the obligate biotrophs: b. graminis   <cit> , although these are up to  <dig> aa in length; p. graminis f. sp.
tritici ; and melampsora larici-populina   <cit> .
these studies use different criteria for ssp identification, hampering valid comparsions.
in the current study, ssps from p. nodorum, c. fulvum and p. graminis f. sp.
tritici were predicted using the same pipeline as that used for ssp analysis from the venturia pathogens.
the number of ssps in the venturia pathogens were between that recorded for the obligate biotroph p. graminis f. sp.
tritici and the necrotroph p. nodorum or the facultative biotroph c. fulvum.
for those ssps predicted to be less than  <dig> aa in length, the proportion predicted to be classically secreted predominated.
this profile resembled that of p. graminis f. sp.
tritici.
thus overall the repertoire of ssps from the venturia pathogens more closely resembles that of an obligate biotroph rather than a necrotroph or facultative biotroph.
evidence of high expression or up-regulation during infection  was used to prioritise a smaller set of venturia ssps  <cit> .
the smallest of these uncharacterised ssps were analysed in more detail and of those with similarity to predicted proteins in related dothideomycete genomes, only four were found in the majority of genomes investigated.
these dothideomycete genomes are from organisms with diverse lifestyles.
up until recently this broad conservation would indicate a core metabolic role, rather than a role in pathogenicity, especially for those proteins also found in the extremophilic sooty mould saprobe, baudoinia compniacensis, with its compact genome of  <dig>  mb  <cit> .
however, the recent research of whigham and associates  <cit>  showing that the broadly conserved effector bec <dig> from the pathogen blumeria, that can suppress hr, may be repurposed to fulfil particular roles in fungi with diverse lifestyles, challenges this view.
a small family of proteins in v. inaequalis with similarity to bys <dig> from m. oryzae also has representatives in other diverse dothideomycetes.
the function of the bys <dig> domain is unknown, but in blastomyces dermatitidis the expression of a bys1-encoding gene is associated with pathogenesis  <cit> , thus a similar role in dothideomycete pathogens cannot be ruled out.
one of the most highly expressed genes in the vis gene set, with expression in vitro and in planta, encodes a protein with similarity to the alternaria alternata major allergen alt a  <dig> .
this v. inaequalis protein was identified previously  <cit>  in an analysis of semi-purified secreted proteins that elicited a response from specific resistant apple hosts.
single genes encoding proteins with similarity to the alt a  <dig> allergen were identified in all venturia genomes and a single gene was also identified in some , related dothideomycetes .
alt a  <dig> is well known in clinical settings as a human allergen.
it has a structure unique to fungi; however, its role in pathogenesis has not as yet been determined  <cit> .
a large number of ssps in the vis set with similar proteins across dothideomycete spp.
have an internal repeat structure.
repeat structures in fungal effectors have been reported previously  <cit> .
for example the sp <dig> effector from glomus intraradices, an arbuscular mycorrhizal biotroph, has nine hydrophilic tandem repeats.
sp <dig> re-programmes plant expression to reduce the defence response thus enabling establishment of the fungus within the roots of plants  <cit> .
in addition, a double knock-out mutant of u. maydis lacking the repeat-containing protein effectors, hum3/rsp <dig>  has arrested growth in planta, shortly after penetration  <cit> .
the roles of the previously identified repeat-containing proteins, cin <dig> and cin <dig>  which are expressed very highly in the stromata and runner hyphae of venturia, are yet to be determined  <cit> .
expanded ssp effector families
intriguingly, venturia strains have multiple genes predicted to encode proteins with similarity to avrlm <dig> from the dothideomycete l. maculans and ave <dig> from the sordariomycete verticillium spp.
these effectors are encoded by a single gene  in verticillium spp.
<cit> , or a single gene plus a paralogue  in l. maculans  <cit> .
the venturia ave1-like genes were generally not found in clusters on contigs and both effector families have significant numbers of pseudogenes .
the role of these ave1-like proteins is yet to be determined; however, the multiple gene members in the avrlm <dig> and ave <dig> effector families will present a challenge for functional analyses.
avrlm6-like genes are not found in most of the sequenced dothideomycete relatives of l. maculans.
the same is true for ave <dig> from the sordariomycete, verticillium.
this discontinuous distribution of ave <dig> and avrlm <dig> suggests that these effectors are either ancient effector ancestors that have been lost or diversified beyond recognition during coevolution and host specialisation, or that there may have been horizontal gene transfer events involving the ave1- and the avrlm6-containing species.
none of these species share a common host, however, and so mechanisms for gene transfer are not obvious.
many of these venturia effector ave <dig> and avrlm <dig> orthologues were not identified by the secretome prediction pipeline as their n-terminus was incorrectly designated by the automated gene calling software packages.
ave1-like proteins have also been detected by mass spectrometry in an analysis of v. pirina proteins expressed in vitro  <cit> .
the presence of a signal peptide, mature n terminus, cleavage site and presence of a conserved intron in the 5′ utr of the gene, was confirmed for several of the v. pirina vp ave1-like genes in this proteogenomic analysis.
the conserved intron in the 5′ utr of ave1-like genes appears to have caused difficulties in accurate gene prediction for all genes in this family.
rigorous interrogation of the whole set of ssps, with a size of  <dig> aa or less, also highlighted the challenges of gene prediction.
on initial comparison of gene predictions, via comparison of their encoded proteins, genes encoding ssps similar to those in the vi <dig> secretome were not predicted in the other genomes.
to verify this absence, comparison of aa sequence against a six-frame translation of the genomes was undertaken and revealed the presence of identical or near-identical loci.
the use of rna-seq data to inform gene prediction in the vi <dig> genome appeared to be more efficient than using a model based upon these data for gene prediction in the other genomes, highlighting the importance of rna-seq data for individual genomes for accurate gene prediction.
with the exception of the families comprising ave1- and avrlm6–like genes, the majority of families of ssp genes in venturia do not have similar genes in the other dothideomycete genomes analysed.
these families appear to be lineage-specific.
lineage-specific genes are common features of fungal genomes sequenced to date  <cit> .
in the venturia pathogens many of the lineage-specific ssps appear to belong to expanded families, for example, eight ssp families with members of less than  <dig> aa have more than  <dig> members, and up to  <dig> members, and are restricted to the venturia genus.
gene gain and also expansion of these lineage-specific families is obviously associated with host range determination and specificity, and the converse, reduction or loss of gene families can likewise be associated with evolution of virulence on a particular host  <cit> .
expansion of lineage-specific effector families has recently been reported in b. graminis  <cit> , with the  <dig> paralogous copies of avr
k <dig> and avr
a <dig>  that contribute to the establishment of the haustorium, being an extreme example  <cit> .
whether the members of the ssp gene families observed in the venturia genomes are derived from a common ancestor remains equivocal since their overall sequence conservation is low.
however, de guillen et al.
<cit>  identified a family of sequence-unrelated, but structurally conserved effectors  in magnaporthe, accounting for 5–10% of the effector repertoire.
these effectors have been presumed to have evolved via diversifying selection rather than convergent evolution  <cit> .
in addition, hydrophobins, with their patchy distribution and low level of sequence conservation, contrasting with a highly conserved structure, related to function, appear to evolve by a birth-and-death mechanism and to be phylogenetically related  <cit> .
the families of venturia ssps with retention of presumably critical cysteines, in terms of structure and stability, may have also arisen under similar evolutionary constraints and thus also be related phylogenetically.
evolution of protein families involves the duplication of an ancestral gene followed by mutation of the duplicated gene to enable novel functionality to emerge  <cit> .
in the case of effectors, expansion and diversification within a family may enable modification of function to prevent alerting a guarding resistance gene product, or structural modification to avoid a direct recognition event that would otherwise elicit a defense response.
thus having expanded families of effectors may offer an evolutionary selective advantage to a pathogen, enabling recognition of an effector to be overcome with evolution of a novel paralogue and deletion or pseudogenization of the previously recognised effector.
the processes involved in how these families expand is still open to debate.
the presence of large numbers of presumably structurally related, but sequence diverse, proteins poses problems for the delineation of families.
indeed, three families were delineated in this study that shared common members.
although an unorthodox compromise, these families were deemed to be too diverse to be amalgamated using the thresholds adopted for this analysis, but the members are sufficiently closely related to form a single family if the thresholds were to be slightly relaxed, thereby highlighting the pitfalls of depending on arbitrary thresholds for either separating or grouping proteins.
in many pathogens, effector genes are associated with tes.
association of tes is evident with the co-evolution of the b. graminis avr
k <dig> effector family with a class of line- <dig> retrotransposons  <cit> , the b. graminis avrpm3a2/f <dig> effector family with various tes  <cit>  and the association of miniature impala transposons  in the promoters of effectors in f. oxysporum  <cit> .
tes have also been implicated in horizontal gene transfer  <cit> .
the analyses of gene density, and association of effector candidates with repeat elements in vi <dig>  suggests that effectors and tes may be similarly associated in venturia.
in addition, most of the avrlm6- and ave1-like genes are closely associated with repeats, i.e.
tes and te-like remnants, as identified by repet  <cit> .
oliver  <cit>  outlined the mutagenic potential of transposons and their, at first, seemingly unlikely contribution to the evolution of a successful pathogen: transposons contribute to diversity generation by insertion either in or near a gene, affecting either structure of the resulting encoded protein or the expression pattern, respectively.
transposons may facilitate gene family expansion through capture and translocation of host genes  <cit> , such expansions can become targets for repeat induced point mutation , with mutations occuring not only in the duplicated transposons but the associated genes  <cit> .
this is an ongoing area of interest and analysis of repeat regions is currently underway in cross progeny of v. inaequalis.
further analyses are required in the venturia spp.
to reveal if tes and indeed rip are involved in diversity generation, and expansion of gene families as suggested by this initial analysis.
CONCLUSIONS
the comparative analysis of whole secreteomes of multiple races of v. inaequalis with the related scab pathogen, v. pirina, has provided novel insights into the unusual, biotrophic lifestyle niche that these pathogens occupy.
it has also yielded significant leads in the hunt for cultivar- and host-specificity determinants of scab fungi.
the challenge now will be to prioritise leads, from the expanded arrays of putative effectors, for futher investigation with a mind to delivering durable, scab control.

 plant-pathogen interactions are a fine interplay between prospective host and pathogen, involving the exchange of molecular signals that determine the outcome of the interaction. the plant endeavours to detect invaders by deploying, as a first line of defence, pattern recognition receptors that recognise pathogen-associated molecular patterns shared by a wide range of non-specialised microbes, with a resulting induction of a low-level defence response termed pamp-triggered immunity. in response, pathogens evolve effectors that enhance a pathogen’s ability to cause disease, often by blocking or suppressing pti. plants in turn evolve resistance proteins that recognise a subset of these effectors either directly or by their actions on plant host targets. thus the respective effector and r protein complements can contribute to the determination of host-range for any given pathogen. the genus venturia belongs to the order venturiales, which is assigned to the class dothideomycetes. the dothideomycetes include many highly destructive plant pathogens, the venturiales being no exception. venturia pathogens are relatively host-specific and infect selected members of the family rosaceae, perhaps the best known and most widely researched of which is v. inaequalis cooke that causes the economically important disease apple scab. related species, for example v. pirina, v. nashicola, v. carpophila and v. cerasi, cause scab diseases of other rosaceae hosts: european pear, asian pear, peach and cherry respectively. venturia species cause similar symptoms by adopting analogous biotrophic parasitic strategies. during infection they penetrate the host cuticle directly and then develop stromata in the cuticle and sub-cuticular space without penetrating host cells. ultimately conidiophores and conidia differentiate from stromata and erupt through the cuticle resulting in the formation of dark, crusty, dry lesions on leaves and fruit. a fundamental question arises as to what determines the host range of these pathogens, especially given that they have similar modes of biotrophic parasitism and infect closely related host species. a further level of complexity underlies pathogen/host species specificity in scab fungi, for example, certain isolates classified as v. inaequalis on the basis of morphological and molecular criteria, as well as their ability to mate, are unable to infect malus, but instead infect different rosaceous hosts, such as eriobotrya and pyracantha. whether these isolates should be considered as separate species or formae speciales is still open to debate. gladieux and associates suggested that there should be no sub-species delineation based on analysis of six nuclear loci. these isolates are therefore very closely related phylogenetically, but have distinct host specificities. host cultivar specificity has long been demonstrated in isolates of v. inaequalis that infect malus; gene-for-gene pairings have been identified to date between races of v. inaequalis and cultivars of malus. only two scab r gene loci in apple have been fully characterised; however, no effectors of v. inaequalis have been characterised to date. work to identify the v. inaequalis effector repertoire that determines cultivar specificity, and most probably host specificity, has been impeded by the lack of a whole genome sequence for this species. genome estimates for v. inaequalis range from 38 mb to 100 mb. the de novo transcriptome of a single isolate of v. inaequalis has been published, with analysis of both in vitro and in planta transcripts. in our study the whole genome sequences of four isolates of v. inaequalis were sequenced using illumina sequencing technologies. the v. inaequalis genomes were compared to a single v. pirina genome. given the extracellular in planta niche the venturia fungi occupy during parasitism it can be assumed that the majority of the factors that enable successful parasitism are first secreted into the sub-cuticular plant-pathogen interface. therefore the secretomes of these isolates are of utmost relevance and so were compared to identify putative pathogencity or virulence factors that may have a role in host determination, with special emphasis on secreted enzymes and putative proteinaceous effectors.  whole genome assemblies and gene predictions a total of 40 mb to 61 mb was assembled for malus-infecting v. inaequalis isolates. the assembled genome size for the loquat scab pathogen was and 41 mb for the pear scab pathogen. lack of sequencing of mate-pair libraries with long inserts contributed to the vi. assembly having the lowest n value among the five isolates, while vp had the largest n. a summary of the repeat content masked for each genome assembly is presented in table  and additional file table the whole genome assemblies of isolates of venturia inaequalis and v. pirina venturia inaequalis venturia pirina malus × domestica malus × domestica malus × domestica eriobotrya japonica pyrus communis number of predicted genesa percentage completeness: partial b percentage completeness: partial c ahybrid with hints/ab initio gene predictions for vi1 bcegma analysis using both partial and full gene sequences cbusco analysis using both partial and full gene sequences dnote the recent race designation change reported for eu-b, previously reported as race by bus et al. gene models were predicted with augustus gene prediction software. the number of gene models in the v. inaequalis isolates ranged from to whereas there were identified in v. pirina. to estimate the completeness of the assembled genomes, the assemblies were scanned for the most highly conserved core eukaryotic genes using the core eukaryotic genes mapping approach. comparisons of partial or complete predicted translated proteins demonstrated that the v. inaequalis vi genome is the most complete, with only one or two core eukaryotic genes missing respectively. the lowest representation of the core eukaryotic genes in any genome was still high, complete and another seven partial predicted proteins for v. inaequalis vil, indicating a reasonably complete whole genome sequence for all isolates. in addition, a benchmarking universal single-copy orthologs analysis was also conducted using busco_v b. this analysis indicated that the v. pirina genome is the most complete and of the v. inaequalis genomes vi and vil are the most complete when partial predicted proteins are considered, although on comparison of complete predicted proteins the vil genome is the most complete. since vi was the most comprehensive v. inaequalis genome in terms of coverage when cegma and busco analyses were taken together, and is predicted to have the most complete set of avirulence effectors, this isolate was selected for preliminary expression analyses. the predicted secretomes of venturia inaequalis and v. pirina to predict the secretome of each isolate, gene models were analysed with a pipeline of programmes. the total number of genes encoding the predicted secretome for each genome is as follows: in vi1; in vi; in vi.; in vil and in vp. the number of predicted proteins in the vi secretome is greater than that for the other genomes due to use of both ab initio and hybrid gene predictions in the analysis. thakur et al. reported a lower predicted secretome number of however this was based on transcriptome data alone and, as such, direct comparison is problematic. gene ontology analysis through an in-house annotation pipeline was used to annotate predicted genes in each secretome; however, the majority of the predicted proteins had no gene ontology classification. hence, for this reason, the carbohydrate active enzyme analysis toolkit, and similarity searches utilising blastp against the ncbi non-redundant database were used in addition to gene ontology. where conflicting designations occurred, cat, then gene ontology took precedence over similarity search designations. the secretome repertoire, in terms of diversity and abundance of protein classes, was similar between all venturia isolates, with small, secreted, non-enzymatic proteins dominating. for comparative purposes the secretomes of three plant pathogenic fungi, parastagonospora nodorum, cladosporium fulvum and puccinia graminis f. sp tritici were also identified using the same pipeline of programmes using predicted proteins downloaded from the joint genome institute. the size of the p. graminis f. sp tritici secretome was significantly larger than that recorded for c. fulvum and p. nodorum. the size of the vi secretome was similar to that of p. graminis f. sp. tritici, whereas the remaining venturia secretomes were of a similar size to those of p. nodorum and c. fulvum.fig. the secretomes of four isolates of v. inaequalis and one isolate of v. pirina. annotations were based on gene ontology analysis including interrogation of ncbi refseq, interpro, uniref, expasy uniprotkb/swiss-prot and expasy prosite, cazyme identification using the cat server and blastp searches against the ncbi non-redundant database. small secreted proteins include predicted proteins with similarity to known effectors and proteins with no known function, either with or without putative conserved motifs identified by pfamscan orthomcl was conducted to assess similarity between the predicted venturia secretomes. a total of isolate-specific singletons were identified but are not included in the orthomcl clustering figure. the venturia core secretome consisted of orthologous clusters, with cazymes and non-enzymatic ssps being the predominant classes. the venturia pan secretome totalled representing individual proteins, with many lineage-specific and host-specificity candidates identified, most of which were predicted to encode non-enzymatic ssps. the majority of these ssps had no significant similarity to proteins in the public domain. the v. inaequalis-unique pan secretome totalled representing individual proteins, with a total of representing individual proteins specific to apple-infecting isolates, with of the clusters found in all three isolates. there were singletons, but no unique clusters specific to the loquat-infecting isolate vil. the v. pirina-specific secretome totalled representing individual proteins, with unique protein clusters and singletons.fig. proteins in the secretomes of four isolates of venturia inaequalis and one of v. pirina. similar proteins in each of the secretomes were identified by orthomcl-. together with the markov clustering algorithm mcl-09-. similarity levels were calculated based on reciprocal blastp similarity searches between the protein sequences with an e value threshold of 1e- figures within the boxes represent the number of proteins in each cluster, whereas figures outside the wheel are the number of clusters in each sector. those proteins that are singletons within each secretome are not represented two sets of rna-seq data, from libraries made with rna harvested from v. inaequalis vi grown in vitro and vi1-infected apple seedling leaves, provided evidence of transcription for % of the vi secretome. the rna-seq data were also used to select a smaller subgroup of the vi secretome to analyse in greater detail. these data were used as indicators of involvement in virulence or pathogenicity. figure  shows representative stages of this material -inoculated apple leaves, as assessed by microscopic evaluation). two approaches were used: first, overall vi gene expression levels were estimated by analysing fragments per kilobase of exon per million reads mapped values. this enabled a ranking of the genes in terms of overall expression during infection to be calculated at both two and seven dpi and the most highly expressed genes at either time point were selected to give the top 5% data set. of the genes identified, were expressed at two and seven dpi, at two only and at seven dpi only. in addition, a differential expression analysis detected of the genes that were up-regulated during growth in planta, compared with in vitro growth. thirty-seven genes were in both the top 5% data set and up-regulated during infection gene sets, giving a total of individual genes.fig. microscopic evaluation of venturia inaequalis vi infection of susceptible apple leaves. a two, and b seven days post inoculation, and c in vitro showing stromatic growth habit fig. predicted proteins present in the vis set from the secretome of venturia inaequalis isolate vi annotations were based on gene ontology analysis including interrogation of ncbi refseq, interpro, uniref, expasy uniprotkb/swiss-prot and expasy prosite, cazyme identification using the cat server and blastp searches against the ncbi non-redundant database. small secreted proteins include predicted proteins with similarity to known effectors and proteins with no known function, either with or without putative conserved motifs identified by pfamscan. the order of the categories in the legend is the same as that in the chart carbohydrate-active enzymes and putative non-cazyme cell wall degrading enzymes the cazyme repertoire was similar between all venturia secretomes, with belonging to the core secretome. of these, all isolates have between and proteins with multiple cazyme domains. proteins were identified as being associated with carbohydrate-binding motif, carbohydrate esterase, glycoside hydrolase, polysaccharide lyase and glycosyl transferase domains. of these, the classes with the greatest number of protein members across each genome were those with: cbm cbm and cbm domains, with specificity for cell wall carbohydrates, and in terms of the latter two, plant cell wall specifically; ce domains with specificity for cutin; gh with specificity for plant cell wall pectin; gh and gh with specificity for β-glycans; and gh with specificity for pectin and hemicelluloses. also identified, although not as numerous, were cazyme domain-containing proteins predicted to interact with fungal cell wall components including: ghs associated with β-glycans, α-glucan, chitin, and cbm domains specific for chitin. the majority of the cazymes were either present in orthomcl clusters that had a single enzyme from each isolate and were thus members of the core secretome set, or in clusters comprised of enzymes from only the four isolates of v. inaequalis, not v. pirina. similar numbers of predicted proteins were identified in the secretome sets, which were similar to enzymes that degrade non-carbohydrate cell-wall components, e.g. ligninases. peptidases and lipases depending on the isolate, to predicted proteins were identified in the venturia secretomes with similarity to peptidases. the majority of these were conserved in the core secretome or in isolates of v. inaequalis rather than v. pirina. there were proteins predicted in the secretomes of only the v. inaequalis isolates that were similar to avr-pita, the avirulence protein from magnaporthe with zinc metalloprotease features that binds directly to its cognate r protein, albeit with a relatively high similarity score of 8e- recorded when the pathogen host interactions database was interrogated. in addition, a further similar protein was also encoded by the genome of v. pirina but was not predicted to be secreted. there were also between eight and proteins, depending on secretome, with similarity to proteases from candida and coccidioides following interrogation of protein sequences against phi-base. predicted proteins with similarity to lipases were also identified in each secretome. primary metabolism and putative proteins involved in redox reactions proteins with a wide range of functionality in primary metabolism, e.g. proteins involved in amino acid or nucleotide metabolism; phosphatases and nitrilases, and unclassified enzymes with an alpha/beta hydrolase fold were identified. depending on the isolate, there were between and proteins in this category in each secretome, most of which were in the core venturia secretome. numerous proteins with putative functions in redox reactions were identified in the venturia secretomes. putative proteins associated with the cell wall, transport, signalling and secondary metabolism as expected there were few proteins in the secretomes associated with the cell wall, transport, signalling and secondary metabolism, since these proteins would be eliminated in the secretome prediction pipeline. the majority of these proteins were members of the core secretome set. small secreted proteins proteins classified as ssps made up between and 66% of each secretome. for comparative purposes, three secretomes derived from publicly available genomes were also screened for ssps using the same pipeline of programmes as those used to analyse the venturia secretomes. ssps made up 79% of the secretome of p. graminis f. sp. tritici, and and 28% of the secretomes of p. nodorum and c. fulvum respectively. smaller ssps less than aa in length predominated in the venturia and p. graminis f. sp. tritici secretomes, especially those with a predicted signal peptide. in contrast, ssps of less than aa in length in the predicted ssp repertoires of c. fulvum and p. nodorum were dominated by those predicted to be secreted by a non-classical mechanism. the majority of genes encoding ssps in vi were supported with evidence of transcription. most ssps are novel to venturia and most of these novel ssps were ≤ aa in length. the vast majority of vis genes, 245/ were predicted to encode ssps, of which were ≤ aa in length. of these smaller ssps, have two or more cysteines in the mature predicted protein. larger secreted proteins with no similarity to known proteins were also represented in each of the secretomes. candidate effectors from the ssp set the secretomes of venturia have several proteins with a putative role in binding hydrophobic surfaces including between and proteins with hydrophobic surface binding domains as identified by pfamscan, with similarity to the aspergillus oryzae hsba, hydrophobic surface binding, cell wall galactomannoprotein. five putative hsba-like genes were identified amongst the vi vis gene set. another gene up-regulated at seven dpi encodes a small predicted mature protein of aa with four cysteines and shares predicted aa sequence identity to bacterial chaplins, which are membrane-associated proteins known to have similar functions to hydrophobins. predicted proteins with similarity to bacterial chaplins are also present in vi, vil and vp, but not vi.. two or three proteins with similarity to fungal hydrophobins were identified in the secretomes of v. inaequalis isolates and four in the v. pirina secretome, on the basis of conserved cysteine patterns. none were included in the vis gene set. the venturia secretomes included predicted ssps with similarity to known effectors from other fungi, including ecp from c. fulvum, avrlm from leptosphaeria maculans and ave from verticillium spp.. the venturia candidate effectors were most similar to these effectors in a reciprocal blast of the cladosporium, leptosphaeria and verticillium genomes. in addition to the orthomcl clustering analysis, further similarity searches within genomes were employed with a more relaxed threshold of 1e- to ascertain whether putative paralogues were present. single orthologues of ecp were identified in each of the venturia genomes analysed, corroborating the data of thakur et al. who reported an orthologue of ecp in the transcriptome of a single apple-specific isolate of v. inaequalis. a search of the pfam database with the vi orthologue of ecp revealed three peptidoglycan-binding, lysin motif domains. at the aa level there is 42% identity between ecp and the v. inaequalis orthologues, whereas the v. pirina orthologue has slightly higher identity at 44%. the vi and vil predicted proteins are identical, whereas that from vi. has one aa substitution compared with the vi1/vil sequence and that from vi has four aa substitutions. the vi ecp orthologue was expressed during growth in planta and in vitro, although expression was not sufficiently high to be in the vis set. orthologues of avrlm and ave are present as large gene families in all of the venturia genomes examined. the copy number of full length avrlm6-like genes varied among v. inaequalis isolates, while the v. pirina isolate had copies. further characterisation of the venturia avrlm6-like gene families is reported in shiller et al.. three avrlm6-like genes are present in the vis gene set. ten full-length ave1-like genes and seven ave1-like pseudogenes, were identified in the whole genome sequence of v. inaequalis vi with similar numbers in all other venturia genomes. the v. inaequalis ave1-like full length, predicted proteins range in similarity from to 57% identity to v. dahliae ave all but one of the vi ave1-like proteins have transcript evidence, with expression at all growth stages, including in vitro, with some very highly expressed during both infection time points. two ave1-like gene loci are represented in the vis gene set. loci of all of the previously identified v. i naequalis candidate effector genes from vi were identified in the vi wgs. however, the majority of the predicted gene models associated with these loci were longer than those identified using ests. one vice gene did not correspond with any augustus gene prediction. only six vice predicted proteins were included in the secretome of vi, five of which were in orthomcl clusters in the core secretome, with a single predicted protein from each isolate in each cluster. the sixth predicted protein belongs to a cluster with members from only vi and vp. genes encoding four of these proteins were in the vis set, including the vice gene. the remaining nine vice gene models, assigned by augustus, did not encode proteins that were predicted to be secreted. inspection of these loci revealed that, in the majority of these cases, the n-terminus predicted by augustus was upstream of the translation start site predicted by bowen and associates. thus the gene predictions at these loci need further validation by sequencing full length cdnas. most of the vice gene transcripts were also detected by thakur et al., in a single transcriptome of v. inaequalis; however, these authors did not confirm whether these transcripts encoded predicted secreted proteins. two cellophane-induced genes, cin and cin that encode proteins with internal repeats, have been previously identified as being highly expressed during both in vitro growth in cellophane membranes and infection of vi on apple. the rna-seq data supported the expression levels reported in the previous study for both of these genes. there were cin orthologues in each of the isolates examined forming a single orthomcl cluster, whereas cin orthologues were only in v. inaequalis isolates, again forming a single orthomcl cluster. cin was one of the most highly expressed genes in the vis gene set, with very high expression in vitro as well as at both infection time points. two additional genes, which encode smaller cysteine-rich cin1-like proteins, cin1l and cin1l, are also found in each of the isolates. however, their sequence is more variable since a single cluster with representative proteins from each isolate was not observed. the proteins similar to cin1l from each v. inaequalis isolate clustered together, but that from the v. pirina isolate was classified as a singleton. there are proteins similar to cin1l in each isolate; however, those proteins encoded by the vi and vi. genomes, although having a signal peptide, were classified as being targeted to the mitochondria by both protcomp and wolf psort, and the proteins from the remaining isolates formed a single cluster. cin1l and cin1l are in the vis gene set. of those proteins originally classified as ssps in the venturia secretomes four shared similarity to proteins in phi-base: the hydrophobin mhp from magnaporthe oryzae; npp from hyaloperonospora arabidopsidis; gas from magnaporthe oryzae; bec from blumeria graminis f. sp. hordei. none of these proteins were members of the vis set, although all had evidence of transcription in vitro and in planta. ssp families in the vis set of the vis ssps ≤ aa in length in the vi secretome, appear to be single proteins following spectral clustering analysis, belong to different families, having between two and members. the families include predicted proteins without in planta up-regulated expression of their corresponding genes and putative proteins that were not predicted by the original gene prediction software. the remaining ssps were not retained in the dataset since, although initially classified as an ssp ≤ aa in length, they belong to families where the majority of members have a robust annotation to an enzyme based on gene ontology analysis and similarity searches, or are larger than aa in length. family and are very closely related and as an indication of this these families share members. a representative sequence logo for family seven is shown in additional file although sequence diverse, the families have a putative conserved structure based on conservation of cysteines.fig. small secreted proteins in the venturia inaequalis vi secretome encoded by single genes. only ssps ≤ amino acids in length are included. the number of similar proteins in the venturia and related dothideomycete proteomes are indicated by numbers: black indicates a gene predicted by augustus, white indicates a putative coding sequence identified by tblastn using the protein sequence as query, followed by manual curation. percentage identity is represented by: red = 100%; orange = 90–99%; yellow = 70–89%; green = 50–69; blue = 30–49% fig. small secreted protein families in four venturia inaequalis and one v. pirina secretomes. only ssps ≤ amino acids in length are included. red = proteins in the same family from vi1; yellow = protein families from vi; green = families from vi.; blue = families from vil; purple = families from vp. families f1-f and f34-f are cysteine rich i.e. two or more cysteines per protein; f29-f are those with one or no cysteines. ★ = families with predicted similar proteins in related dothideomycete genomes thirty families of predicted ssps, and single proteins, have similar proteins present in all of the venturia isolates analysed. all, except five, of the single proteins are also single proteins in all of the isolates studied. seven families are unique to and conserved within the isolates of v. inaequalis, whereas there are single proteins in vi with similar proteins present in all v. inaequalis isolates and absent from v. pirina. there is one family, and two single proteins, restricted to isolates that only infect apple. in addition, there are four single ssp proteins, and one family, that are putative candidates for the effector/s that contribute to the race profile of isolate vi., i.e. present in vi and vi, but absent from vi.. there is one family and three single proteins that are candidates for effector avrrvi given their presence and absence in the venturia isolates.table small secreted proteins in the venturia infection secretome set and related fungi both families and single genes encoding small secreted proteins ≤ amino acids in length are included. families were analysed by spectral clustering followed by manual curation and analysis of non-predicted putative genes aticks in columns indicate presence of a similar protein or proteins in that secretome to ascertain whether any of the ssps of the vis set were conserved across the dothideomycetes, similarity searches were made against selected dothideomycete predicted proteomes curated at the jgi; this curation includes clustering information pertaining to proteins from each organism. only ssps predicted for the vi secretome, which were present in all the venturia isolates, had putative similar members in related dothideomycete predicted proteomes.fig. similar small secreted proteins in dothideomycete proteomes and the venturia inaequalis vi secretome. only ssps ≤ amino acids in length are included. shades of blue, purple and pink = pleosporales: cochliobolus sativus, c. heterostrophus c c. heterostrophus c c. lunatus, c. miyabeanus, c. victoriae, pyrenophora tritici-repentis, p. teres f. teres, leptosphaeria maculans, parastagonospora nodorum; shades of orange, yellow and red = capnodiales: septoria populicola, s. musiva, m. fijiensis, cladosporium fulvum, dothistroma septosporum, zymoseptoria tritici, baudoinia compniacensis; green = dothideales: aureobasidium pullulans var. pullulans putative host range determinants in the ssp set in addition to those candidate effectors that are specific to either v. inaequalis, or those isolates able to infect apple described above, ssps ≤ aa in length specific to the loquat-infecting isolate of v. inaequalis or to v. pirina were identified by orthomcl analysis. in addition to the candidate effectors specific to vil, only four of which had any similarity to proteins in the ncbi nr database, were found in both the vil and vp secretomes. a further candidate effectors were found to be specific to v. pirina. gene density and proximity of genes to transposable elements most genes in the vi genome have intergenic regions between 200 bp and 3 kb, with a mean intergenic distance of 1677 bp. a subset of core eukaryotic genes had a slightly shorter mean intergenic distance of 1289 bp, whereas the ssp genes in the vis set, display a significantly longer average intergenic distance at 2560 bp. genes located on scaffold ends, hence lacking neighbouring genes, were excluded from this analysis. this excluded 17/ from the core eukaryotic gene set and 21/ from the ssps in the vis set. the greater proportion of ssp genes located on the ends of scaffolds suggests a bias for association with complex repeat regions that cause problems with sequencing and assembly, resulting in broken genomic scaffolds.fig. flanking distance between predicted genes of venturia inaequalis vi intergenic distances for all predicted genes are represented in the underlying heatmap, with the number of genes in each bin shown as a colour-coded heat map generated as in saunders et al.. genes were sorted into two-dimensional bins on the basis of the lengths of flanking intergenic distances to neighbouring genes at their 5′ and 3′ ends; overlying this are scatterplots of a core eukaryotic genes or b venturia infection secretome gene set, plus avrlm6- and ave1-like genes; light pink = ssps with one or no cysteines; blue = peptidases; dark green = cazymes; light green = putative cell wall-degrading enzymes; white = cell wall associated and miscellaneous proteins > amino acids). note that the axes are not linear. genes at the scaffold end were excluded from this analysis concordantly, ssp genes also appear to be more closely associated with transposable elements or te-like features than core eukaryotic genes and all remaining genes in the vi genome. the mean gene to te distance was 9024 bp for all genes predicted in the genome. the core eukaryotic gene set had a significantly greater mean distance to the nearest te of  bp, whereas the ssps in the vis set had a mean distance to the nearest te of only 5401 bp. twenty-two ssps in the vis set were nested within predicted te-like features. fourteen of the core eukaryotic genes and six of the vis set ssp genes were excluded from this analysis as they lacked a neighbouring te feature on the same scaffold.fig. distance between predicted genes and transposable element -like features of venturia inaequalis vi flanking distances to te-like features for all predicted genes are represented in the underlying heat map, with the number of genes in each bin shown as a colour-coded heat map on orthogonal projection. genes were sorted into two dimensional bins on the basis of the lengths of flanking distances. a core eukaryotic genes are similar to all genes in that they are not closely associated with te-like features; whereas b venturia infection secretome gene set, plus avrlm6- and ave1-like genes; light pink = ssps with one or no cysteines or less; blue = enzymes; dark green = cazymes; light green = putative cell wall-degrading enzymes; white = cell wall associated and miscellaneous proteins > amino acids in length). note that the axes are not linear. genes at the scaffold end were excluded from this analysis discussion comparisons of whole genome assemblies and gene predictions this paper reports the first analysis of whole genome sequences from multiple isolates of v. inaequalis. the sizes of all five venturia genomes are comparable to those of other dothideomycetes; however, these may be an underestimate since the majority of the sequencing was carried out on the illumina platform. assembling repeat regions using short reads is notoriously difficult, and hinders genome assembly using de bruijn graph-based genome assemblers like velvet and allpaths-lg. in addition, there is an apparent wide range of genome size that can be largely attributed to additional repetitive content, with the assembled genic regions being similar in size for all five venturia isolates. whilst variation in genome size between isolates from the same species is not unprecedented; for example, genome size in three isolates of the dothideomycete zymoseptoria tritici ranged from to 40 mb as estimated by pulsed field gel electrophoresis, this apparent variation in the venturia genomes may be attributable to the different strategies, and software, used for sequencing. the genomes assembled using allpaths-lg software, have much larger genome sizes and higher percentages of repeats, than those assembled using velvet. the major difference between the sequencing of these five isolates is that vi and vil paired-end libraries were specifically constructed with an insert size of 180 bp so that paired reads overlapped at the end, to produce longer super-reads. for the other three isolates, the pe libraries were constructed with a typical insert size of 400 bp, and paired reads did not overlap and could not be assembled using allpaths-lg. the super-reads generated by allpaths-lg result in repeats being better assembled. as a trial, in addition to allpaths-lg, the vi and vil sequences were also assembled using velvet. the percentage of repeats in the velvet scaffolds for each isolate was lower than that for the allpaths-lg assembly, but was still very high compared with the remaining three isolates, with and 25% for vi and vil respectively. additional investigation is required to determine whether the larger genome sizes and higher percentages of repeats in vi and vil are a biological reality. the number of predicted genes in the venturia genomes is significantly less than the unique fungal genes reported for v. inaequalis by thakur et al.. just over half of the reported sequences had no significant similarity to sequences in other species. the thakur et al. estimate was based on in vitro and in planta transcriptome data only, with multiple gene splice variants likely to account for the higher gene number. our estimate of predicted gene number is comparable to related dothideomycete genomes which range from in s. populicola to in c. fulvum. gene number variation could be due to difference in coverage of the various genomes; however, variation in gene number between isolates is also not unprecedented. for example, xue and associates found that in the rice blast fungus m. oryzae, gene number varied, with hundreds of isolate-specific genes present in genomes of field isolates. similar gene number variation was reported in isolates of c. heterostrophus. venturia pathogens: lifestyle and host determination focussing on the secretome of the venturia pathogens has revealed repertoires of proteins that reflect aspects of their adopted mode of parasitism, with secretion of compounds to adhere to the waxy, water-proof cuticle on leaves and fruit and to enable direct penetration and colonisation of the cuticle and sub-cuticular space. in addition, effectors that are predicted to effect evasion of recognition and suppression of host defence will be secreted to the extracellular plant-pathogen interface, including those that may be directed to be taken up by the host cells. attachment and penetration several venturia proteins were identified in the secretomes that may be involved in attachment and penetration of the cuticle. a predicted protein present in all the dothideomycete genomes analysed has similarity to a m. oryzae fasciclin protein. fasciclins have been implicated in cell adhesion in diverse organisms including both prokaryotes and eukaryotes. the m. oryzae fasciclin has an important role in development and pathogenicity, involved in conidiation and conidial adhesion. a similar role for fasciclins in cell adhesion for all the venturia isolates analysed is easily envisaged. hydrophobins are small, secreted, cysteine-rich, amphipathic proteins that are usually found on the cell walls of fungi. they provide a water-repellent coat and are well characterized for their role in morphogenesis and virulence in plant pathogenic fungi, promoting interactions with hydrophobic surfaces. it is thought that these proteins may also assist in avoiding detection by the host during infection. hydrophobins are usually highly variable in sequence; however, they have a conserved structure due to conserved cysteine residues. venturia secretomes have fewer hydrophobin genes than c. fulvum but have a similar number to d. septosporum. the venturia secretomes also have a high number of genes encoding putative hsba proteins. hsba proteins are found in entomopathogenic fungi as well as pathogens of higher animals; with cuticle colonisation being a possible common link. hsbas are not commonly reported to be encoded by plant pathogen genomes. one sequence with similarity to an hsba was identified in the genome of the mesophyll apoplast-dwelling c. fulvum and the pine pathogen d. septosporum. hsbas are not hydrophobins, but they have been reported to have an analagous role in a. oryzae in binding to hydrophobic surfaces and recruiting the cutl polyesterase/cutinase to degrade the polyester substrate, poly. takahashi and associates also reported that the a. oryzae hydrophobin, rola, also recruits cutl to aid degradation of pbsa surfaces. thus, a. oryzae appears to use several types of proteins to recruit lytic enzymes to the surface of hydrophobic solid materials and promote their degradation. proteins similar to the a. oryzae cutl polyesterase/cutinase and hsba were well represented in the vis set. extracellular cutinase has been implicated in penetration by v. inaequalis, as cutinase is produced by germinating conidia, and a cutinase inhibitor can prevent penetration. in addition, esterase-like activity has been reported during the germination of conidia and in appressoria. the presence of high numbers of ces in the venturia secretomes supports the experimental evidence of enzymatic penetration of the cuticle by v. inaequalis and the inference that v. pirina also may act similarly. we propose that venturia, like a. oryzae, uses multiple proteins to recruit cutinases/esterases to facilitate appressorial adhesion and direct penetration, as well as degradation and digestion of cuticle. this hypothesis fits observations of the cuticular degradation that occurs during colonisation by venturia fungi potentially drawing nutrition from the cuticle or cuticle precursors. nutrition exploitation of the cuticle may be insufficient to satisfy pathogen nutritional requirements throughout the infection cycle. during biotrophic infection venturia remains in the cuticle and sub-cuticular space causing relatively little damage to the host. most damage is due to breaching of the cuticle upon sporulation. at this time the epidermal cells underlying the stroma undergo a progressive depletion of plastids and cytoplasm, accompanied by increasing vacuolation, leading ultimately to cell death. it has been proposed that this damage late in the infection cycle is caused by partial cell wall degradation. indeed, cellulase and pectinase activities have all been reported for v. inaequalis growing in vitro. however, it has been suggested that the timing of this host cell degradation precludes a significant role for these enzymes in nutrient acquisition. the predominant polysaccharide in apple fruit skin is pectin at 65%, compared with a level of 3% for cellulose, with pear fruit also having higher levels of pectin than cellulose. venturia appears to have tailored its cazyme repertoire to suit the composition of the host cell wall with pectin-specific cazymes predominating; the two most numerous classes of cazymes found in the venturia secretomes, after those with cutinase activity, are gh and gh that have pectin as a substrate. a single gh enzyme and two putative pls, that also have pectin as target substrate, are up-regulated during infection and are present in the vis set. significantly, this lytic activity may therefore not be limited to late in the infection cycle as previously thought, but contribute to nutrition via degrading the surface polysaccharides of the epidermal cells beneath stromata, this damage only becoming evident macroscopically late in infection. in addition, those cazymes present in the secretomes of the venturia fungi may also benefit the fungus during saprobic growth and sexual fruiting body development during winter. evasion of host defence the putative proteases present in the secretomes of the venturia fungi may aid in the evasion of host defense since proteases have been implicated in plant defence avoidance in many plant-pathogen interactions. for example, fungal proteases can target plant chitinases, as in the interaction between fusarium oxysporum f. sp lycopersici and tomato where both a serine protease and a metalloprotease are required for the inactivation of a chitin-binding domain -containing chitinase thus contributing to virulence. as a possible alternative strategy to counteract host chitinases the venturia strains, in common with many other fungi, all have a homologue of the c. fulvum lysm domain effector, ecp these are likely to either have a similar function to c. fulvum ecp in preventing recognition of chitin fragments capable of initiating a defence response, or to the lysm effectors from z. tritici that protect hyphae from the action of chitinases, thus preventing the release of chitin-derived pamps. in addition, there are single proteins in each secretome similar to the blumeria effector bec. the putative venturia bec orthologues all have the conserved etvic motif that is required for suppression of the hypersensitive response in barley. a similar function for these proteins during the pathogenicity of venturia is therefore easily envisaged. although single proteins similar to the npp protein from h. arabidopsidis are present in each of the venturia secretomes the sequence of these from two apple-infecting isolates are truncated. in the remaining secretomes the npp1-similar proteins appear to belong to the type i nep-1-like protein family, with the two conserved cysteines. all three proteins also have two additional c-terminal cysteines, however these are not characteristic of type nlps. the majority of nlps induce necrosis in a wide-range of dicotyledonous plants, although whether this necrosis is a direct result of cytotoxicity via dispruption of the plasma membrane or initiation of a defence response remains open to debate. however, the requirement for a functional necrosis-inducing protein in the biotrophic venturia pathogens is unlikely. indeed, each of the venturia proteins has mutations in the critical h residue in the loop region required for necrosis in the nlp from v. dahliae, whilst the two v. inaequalis proteins also have a mutation in the heptapeptide domain required for necrosis in nlps from diverse taxa. these mutations may therefore prevent unwanted pathogen-induced necrosis, but the retention of these nlp-like proteins by venturia may indicate a virulence function that remains to be elucidated. venturia predicted proteins with similarity to the magnaporthe gas protein, required for appressorial penetration and lesion development, may play a similar role in apple and pear scab diseases to that observed in rice. whether gas is translocated to the host cytoplasm with a role in defence suppression remains equivocal, however; weak fluorescence of fluorescent protein constructs in infectious hyphae in onion epidermal cells was observed, as was translocation to rice cytoplasm, albeit under, presumably, the heterologous protomoter from the magnaporthe gene pwl. the venturia proteins may therefore be translocated to the host cell cytoplasm where they may have a role in re-directing host metabolism during scab disease, however this remains conjecture. initiation of host resistance: an avirulence function the comparative approach undertaken here has enabled preliminary identification of candidates for v. inaequalis avirulence effectors avrrvi avrrvi avrrvi and avrrvi which have an avirulence function and are recognised by cognate r proteins. all candidates have cysteine residues but no recognisable protein motifs, except a single candidate for avrrvi avrrvi or avrrvi that is similar to a bacterial chaplin with a domain of unknown function, that may play a role in the venturia lifecycle similar to that of hydrophobins. in addition to ssps that may be involved in race/cultivar specificity on apple within v. inaequalis, further ssps were identified that are specific to the loquat-specific isolate of v. inaequalis or to v. pirina. the majority of these ssps were either similar to hypothetical proteins available in the public domain or had no similarity with known proteins. however, of interest is a protein with ankyrin repeats that may be involved in the specificity of v. pirina, since it is dissimilar to proteins found in the other secretomes. ankyrin repeat-containing proteins are involved in protein-protein interactions and are present in effectors from both prokaryotes and eukaryotes, and as such this protein may play a role in the interaction with european pear. in addition, a single protein specific to the vil isolate has a snoal-like domain. snoal-like domains are found in proteins from diverse taxa, including filamentous fungal phytopathogens. for example, the protein pep from nectria haematococca mpvi has such a domain and contributes to virulence on pea; complementing an isolate of n. haematococca lacking a supernumerary chromosome bearing pathogenicity genes with pep results in a small, but significant, increase in virulence. how these candidate effectors may be contributing to host range determination is unknown. they may be acting as avirulence proteins during attempted infection of a nonhost, with a recognition event triggering resistance, and a concomitant lack of recognition in a compatible host. indeed, since the hosts are closely related, the contribution of r protein-triggered immunity towards nonhost resistance has been postulated to be greater than for host plants that are more distantly related. the variability of the closely related genomes of the venturiaceae with respect to ssps reinforces the utility of a comparative genomic approach. in each of the v. inaequalis secretomes the predicted proteins with similarity to avr-pita from m. oryzae may play a similar role to that adopted by both avr-pita and its paralogue avr-pita in the rice blast interaction. although the virulence function of avr-pita a putative zinc metalloprotease, has not been verified it is directly recognised by the cognate r gene product pi-ta. an avirulence function for the similar proteins in the venturia pathogens remains to be elucidated. effectors with unknown function in addition to those proteins with similarity to known effectors with roles partially or fully confirmed, the venturia secretomes comprise a large repertoire of putative effector ssps with no obvious hints as to function from similarity searches. the paradigm for the role of ssps is that they are lineage-specific effectors that contribute to the maintenance and evolution of parasitism and that obligate biotrophy is associated with expanded effector repertoires. it is thought that biotrophs require a more extensive and nuanced effector inventory than that required by necrotrophs, that rely more prominently on cell-wall degrading enzymes and toxins. although this précis of fungal pathogen lifestyles is highly simplistic, it seems to be reflected in the fewer number of ssps recorded for necrotrophic fungal phytopathogens. for example parastagonospora nodorum has a suite of only ssps, and a recent analysis of the secretome of sclerotinia sclerotiorum revealed only effector candidates, albeit using different criteria for selection. this contrasts with the numbers of ssps reported for the obligate biotrophs: b. graminis, although these are up to aa in length; p. graminis f. sp. tritici; and melampsora larici-populina. these studies use different criteria for ssp identification, hampering valid comparsions. in the current study, ssps from p. nodorum, c. fulvum and p. graminis f. sp. tritici were predicted using the same pipeline as that used for ssp analysis from the venturia pathogens. the number of ssps in the venturia pathogens were between that recorded for the obligate biotroph p. graminis f. sp. tritici and the necrotroph p. nodorum or the facultative biotroph c. fulvum. for those ssps predicted to be less than aa in length, the proportion predicted to be classically secreted predominated. this profile resembled that of p. graminis f. sp. tritici. thus overall the repertoire of ssps from the venturia pathogens more closely resembles that of an obligate biotroph rather than a necrotroph or facultative biotroph. evidence of high expression or up-regulation during infection was used to prioritise a smaller set of venturia ssps. the smallest of these uncharacterised ssps were analysed in more detail and of those with similarity to predicted proteins in related dothideomycete genomes, only four were found in the majority of genomes investigated. these dothideomycete genomes are from organisms with diverse lifestyles. up until recently this broad conservation would indicate a core metabolic role, rather than a role in pathogenicity, especially for those proteins also found in the extremophilic sooty mould saprobe, baudoinia compniacensis, with its compact genome of  mb. however, the recent research of whigham and associates showing that the broadly conserved effector bec from the pathogen blumeria, that can suppress hr, may be repurposed to fulfil particular roles in fungi with diverse lifestyles, challenges this view. a small family of proteins in v. inaequalis with similarity to bys from m. oryzae also has representatives in other diverse dothideomycetes. the function of the bys domain is unknown, but in blastomyces dermatitidis the expression of a bys1-encoding gene is associated with pathogenesis, thus a similar role in dothideomycete pathogens cannot be ruled out. one of the most highly expressed genes in the vis gene set, with expression in vitro and in planta, encodes a protein with similarity to the alternaria alternata major allergen alt a. this v. inaequalis protein was identified previously in an analysis of semi-purified secreted proteins that elicited a response from specific resistant apple hosts. single genes encoding proteins with similarity to the alt a allergen were identified in all venturia genomes and a single gene was also identified in some, related dothideomycetes. alt a is well known in clinical settings as a human allergen. it has a structure unique to fungi; however, its role in pathogenesis has not as yet been determined. a large number of ssps in the vis set with similar proteins across dothideomycete spp. have an internal repeat structure. repeat structures in fungal effectors have been reported previously. for example the sp effector from glomus intraradices, an arbuscular mycorrhizal biotroph, has nine hydrophilic tandem repeats. sp re-programmes plant expression to reduce the defence response thus enabling establishment of the fungus within the roots of plants. in addition, a double knock-out mutant of u. maydis lacking the repeat-containing protein effectors, hum3/rsp has arrested growth in planta, shortly after penetration. the roles of the previously identified repeat-containing proteins, cin and cin which are expressed very highly in the stromata and runner hyphae of venturia, are yet to be determined. expanded ssp effector families intriguingly, venturia strains have multiple genes predicted to encode proteins with similarity to avrlm from the dothideomycete l. maculans and ave from the sordariomycete verticillium spp. these effectors are encoded by a single gene in verticillium spp., or a single gene plus a paralogue in l. maculans. the venturia ave1-like genes were generally not found in clusters on contigs and both effector families have significant numbers of pseudogenes. the role of these ave1-like proteins is yet to be determined; however, the multiple gene members in the avrlm and ave effector families will present a challenge for functional analyses. avrlm6-like genes are not found in most of the sequenced dothideomycete relatives of l. maculans. the same is true for ave from the sordariomycete, verticillium. this discontinuous distribution of ave and avrlm suggests that these effectors are either ancient effector ancestors that have been lost or diversified beyond recognition during coevolution and host specialisation, or that there may have been horizontal gene transfer events involving the ave1- and the avrlm6-containing species. none of these species share a common host, however, and so mechanisms for gene transfer are not obvious. many of these venturia effector ave and avrlm orthologues were not identified by the secretome prediction pipeline as their n-terminus was incorrectly designated by the automated gene calling software packages. ave1-like proteins have also been detected by mass spectrometry in an analysis of v. pirina proteins expressed in vitro. the presence of a signal peptide, mature n terminus, cleavage site and presence of a conserved intron in the 5′ utr of the gene, was confirmed for several of the v. pirina vp ave1-like genes in this proteogenomic analysis. the conserved intron in the 5′ utr of ave1-like genes appears to have caused difficulties in accurate gene prediction for all genes in this family. rigorous interrogation of the whole set of ssps, with a size of aa or less, also highlighted the challenges of gene prediction. on initial comparison of gene predictions, via comparison of their encoded proteins, genes encoding ssps similar to those in the vi secretome were not predicted in the other genomes. to verify this absence, comparison of aa sequence against a six-frame translation of the genomes was undertaken and revealed the presence of identical or near-identical loci. the use of rna-seq data to inform gene prediction in the vi genome appeared to be more efficient than using a model based upon these data for gene prediction in the other genomes, highlighting the importance of rna-seq data for individual genomes for accurate gene prediction. with the exception of the families comprising ave1- and avrlm6–like genes, the majority of families of ssp genes in venturia do not have similar genes in the other dothideomycete genomes analysed. these families appear to be lineage-specific. lineage-specific genes are common features of fungal genomes sequenced to date. in the venturia pathogens many of the lineage-specific ssps appear to belong to expanded families, for example, eight ssp families with members of less than aa have more than members, and up to members, and are restricted to the venturia genus. gene gain and also expansion of these lineage-specific families is obviously associated with host range determination and specificity, and the converse, reduction or loss of gene families can likewise be associated with evolution of virulence on a particular host. expansion of lineage-specific effector families has recently been reported in b. graminis, with the paralogous copies of avr k and avr a that contribute to the establishment of the haustorium, being an extreme example. whether the members of the ssp gene families observed in the venturia genomes are derived from a common ancestor remains equivocal since their overall sequence conservation is low. however, de guillen et al. identified a family of sequence-unrelated, but structurally conserved effectors in magnaporthe, accounting for 5–10% of the effector repertoire. these effectors have been presumed to have evolved via diversifying selection rather than convergent evolution. in addition, hydrophobins, with their patchy distribution and low level of sequence conservation, contrasting with a highly conserved structure, related to function, appear to evolve by a birth-and-death mechanism and to be phylogenetically related. the families of venturia ssps with retention of presumably critical cysteines, in terms of structure and stability, may have also arisen under similar evolutionary constraints and thus also be related phylogenetically. evolution of protein families involves the duplication of an ancestral gene followed by mutation of the duplicated gene to enable novel functionality to emerge. in the case of effectors, expansion and diversification within a family may enable modification of function to prevent alerting a guarding resistance gene product, or structural modification to avoid a direct recognition event that would otherwise elicit a defense response. thus having expanded families of effectors may offer an evolutionary selective advantage to a pathogen, enabling recognition of an effector to be overcome with evolution of a novel paralogue and deletion or pseudogenization of the previously recognised effector. the processes involved in how these families expand is still open to debate. the presence of large numbers of presumably structurally related, but sequence diverse, proteins poses problems for the delineation of families. indeed, three families were delineated in this study that shared common members. although an unorthodox compromise, these families were deemed to be too diverse to be amalgamated using the thresholds adopted for this analysis, but the members are sufficiently closely related to form a single family if the thresholds were to be slightly relaxed, thereby highlighting the pitfalls of depending on arbitrary thresholds for either separating or grouping proteins. in many pathogens, effector genes are associated with tes. association of tes is evident with the co-evolution of the b. graminis avr k effector family with a class of line- retrotransposons, the b. graminis avrpm3a2/f effector family with various tes and the association of miniature impala transposons in the promoters of effectors in f. oxysporum. tes have also been implicated in horizontal gene transfer. the analyses of gene density, and association of effector candidates with repeat elements in vi suggests that effectors and tes may be similarly associated in venturia. in addition, most of the avrlm6- and ave1-like genes are closely associated with repeats, i.e. tes and te-like remnants, as identified by repet. oliver outlined the mutagenic potential of transposons and their, at first, seemingly unlikely contribution to the evolution of a successful pathogen: transposons contribute to diversity generation by insertion either in or near a gene, affecting either structure of the resulting encoded protein or the expression pattern, respectively. transposons may facilitate gene family expansion through capture and translocation of host genes, such expansions can become targets for repeat induced point mutation, with mutations occuring not only in the duplicated transposons but the associated genes. this is an ongoing area of interest and analysis of repeat regions is currently underway in cross progeny of v. inaequalis. further analyses are required in the venturia spp. to reveal if tes and indeed rip are involved in diversity generation, and expansion of gene families as suggested by this initial analysis.  the comparative analysis of whole secreteomes of multiple races of v. inaequalis with the related scab pathogen, v. pirina, has provided novel insights into the unusual, biotrophic lifestyle niche that these pathogens occupy. it has also yielded significant leads in the hunt for cultivar- and host-specificity determinants of scab fungi. the challenge now will be to prioritise leads, from the expanded arrays of putative effectors, for futher investigation with a mind to delivering durable, scab control. 
candidates for avirulence effectors with cognate resistance genes involved in race-cultivar specificity were identified, as were putative proteins involved in host-species determination. genomes of venturia pirina and venturia inaequalis were sequenced and the predicted secretomes of each isolate identified. venturia inaequalisventuria pirinaapplemalus x domesticaeuropean pearpyrus communissecretomeeffectorthe new zealand foundation for research, science and technologyc06x c06x0207victorian life sciences computation initiative’s life sciences computation centrevictorian life sciences computation initiative’s life sciences computation centrehttp://dx.doi.org/ /501100001215la trobe universityvictorian life sciences computation initiativeissue-copyright-statement© the author 2017 in terms of economic impact, the most important of these are v. inaequalis, which infects apple, and v. pirina, which is a pathogen of european pear. candidate effectors were found, on average, to be in regions of relatively low gene-density and in closer proximity to repeats, compared with core eukaryotic genes. given that venturia fungi colonise the sub-cuticular space without penetrating plant cells, it is assumed that effectors that contribute to virulence and determination of host range will be secreted into this plant-pathogen interface. effectors that are putative determinants of host range were identified; both those that may be involved in race-cultivar and host-species specificity. comparative secretomics has revealed candidate effectors from venturia fungal plant pathogens that attack pome fruit. numerous genes with similarity to leptosphaeria maculans avrlm and the verticillium spp. since many of the effector candidates are in close proximity to repetitive sequences this may point to a possible mechanism for the effector gene family expansion observed and a route to diversification via transposition and repeat-induced point mutation. rna-seq was conducted on the apple-specific v. inaequalis isolate vi to highlight virulence and pathogenicity components of the secretome. thus the predicted secretomes of a range of isolates of venturia with distinct host-ranges were interrogated to reveal putative proteins involved in virulence and pathogenicity. fungal plant pathogens belonging to the genus venturia cause damaging scab diseases of members of the rosaceae.
 fungal plant pathogens belonging to the genus venturia cause damaging scab diseases of members of the rosaceae. in terms of economic impact, the most important of these are v. inaequalis, which infects apple, and v. pirina, which is a pathogen of european pear. given that venturia fungi colonise the sub-cuticular space without penetrating plant cells, it is assumed that effectors that contribute to virulence and determination of host range will be secreted into this plant-pathogen interface. thus the predicted secretomes of a range of isolates of venturia with distinct host-ranges were interrogated to reveal putative proteins involved in virulence and pathogenicity.  genomes of venturia pirina and venturia inaequalis were sequenced and the predicted secretomes of each isolate identified. rna-seq was conducted on the apple-specific v. inaequalis isolate vi to highlight virulence and pathogenicity components of the secretome. genes encoding over small secreted proteins were identified, most of which are novel to venturia, with expansion of putative effector families a feature of the genus. numerous genes with similarity to leptosphaeria maculans avrlm and the verticillium spp. ave were identified. candidates for avirulence effectors with cognate resistance genes involved in race-cultivar specificity were identified, as were putative proteins involved in host-species determination. candidate effectors were found, on average, to be in regions of relatively low gene-density and in closer proximity to repeats, compared with core eukaryotic genes.  comparative secretomics has revealed candidate effectors from venturia fungal plant pathogens that attack pome fruit. effectors that are putative determinants of host range were identified; both those that may be involved in race-cultivar and host-species specificity. since many of the effector candidates are in close proximity to repetitive sequences this may point to a possible mechanism for the effector gene family expansion observed and a route to diversification via transposition and repeat-induced point mutation. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords venturia inaequalisventuria pirinaapplemalus x domesticaeuropean pearpyrus communissecretomeeffectorthe new zealand foundation for research, science and technologyc06x c06x0207victorian life sciences computation initiative’s life sciences computation centrevictorian life sciences computation initiative’s life sciences computation centrehttp://dx.doi.org/ /501100001215la trobe universityvictorian life sciences computation initiativeissue-copyright-statement© the author 2017 