BACKGROUND
plant pathogens secrete effectors to influence host metabolism or defense mechanisms to provide an environment for successful infection  <cit> .
effectors can be recognized by plant resistance  genes, eliciting effector-triggered immunity  in the plant to prevent further infection  <cit> .
effectors that are recognized by the plant are known as avirulence  proteins in the context of gene-for-gene interactions.
alteration of avr genes enables parasites to avoid r-dependent recognition and thus overcome host resistance  <cit> .
the avrk <dig> and avra <dig> genes of the barley powdery mildew fungus, blumeria graminis f.sp.
hordei , encode proteins which have a dual role as effectors and as avr proteins, the presence of which is recognized by the mlk <dig> and mla <dig> r-proteins.
these genes were cloned from isolate cc <dig>  by genetic and physical mapping  <cit> .
numerous lines of genetic and biological evidence indicate their dual function.
the avr genes each co-segregate with the respective avirulence phenotype, have a functional open reading frame  in avirulent but not virulent isolates, and are expressed by isolates avirulent to the corresponding r-gene.
the specific recognition of avrk <dig> and avra <dig> by mlk <dig> and mla <dig> r-proteins and subsequent cell death was shown in two independent series of experiments by transient expression of avrk <dig> and avra <dig> in barley plants carrying mlk <dig> or mla <dig> resistance genes  <cit> .
specific recognition of avrk <dig> and avra <dig> also induced inaccessibility to subsequent infection and reduced fungal sporulation  <cit> .
their effector function was demonstrated in two independent experiments.
first, when they were transiently overexpressed in susceptible plants lacking the corresponding r-gene, they increased the infectivity of bgh  <cit> .
second, host-induced gene silencing  of these genes in susceptible plants caused a reduction in haustorium formation by bgh  <cit> .
further evidence for effector and avr functions has been obtained for avra <dig>  this gene is expressed at low levels in conidia that have just landed on the leaf surface, followed by rapid induction within 6 h and high expression until 24 h after inoculation, with a drop thereafter  <cit> .
the interaction between the mla <dig> protein and the wrky <dig> transcription factor depended on the presence of avra <dig> protein for defense gene activation  <cit> .
an ems-induced point mutation of avra <dig>  producing premature termination of the coding sequence, rendered the pathogen virulent to plants carrying the mla <dig> resistance gene   <cit> .
natural bgh isolates virulent to mla <dig> or mlk <dig> do not have functional variants of avrk <dig> or avra <dig> in the corresponding loci, in many cases due to the fusion of the avr genes with retrotransposon sequences  <cit> .
avrk <dig> and avra <dig> are homologues , and are the first discovered members of the eka gene family , with more than  <dig> homologues in the bgh genome  <cit> .
the eka family has no known homologues outside powdery mildew fungi  <cit>  but is present in different formae speciales of blumeria graminis  <cit>  and in powdery mildew species infecting different host such as pea, grape, plantain or arabidopsis thaliana .
a previous analysis showed that these avrk <dig> and avra <dig> homologues are frequently found in the same orf as the nucleotide binding  domain of a line retrotransposon, that the avr-homologous and nb domains are expressed in different bgh isolates as a single transcript, and that both type of sequences have coevolved  <cit> .
line retrotransposons are almost ubiquitous in fungi, plants and animals  <cit> .
they are able to replicate autonomously, and their mobility is dependent on target-primed reverse transcription  <cit> .
these elements typically consist of an orf  containing a gene encoding a reverse transcriptase  and an endonuclease.
in addition, orf <dig>  usually found in the l <dig>  i and jockey groups  <cit> , may have been acquired independently on multiple occasions during the evolution of line elements  <cit> .
the best studied orf <dig> protein  is that in the l <dig> superfamily of line elements.
orf1p is thought to assist l <dig> retrovirus-like particles to gain access to the nucleus, where it can interact with genomic dna and thus initiate integration through target primed reverse transcription  <cit> .
the orf1p of the second major superfamily of lines  is only poorly characterized and its function is not yet fully understood  <cit> .
it usually contains at least one non-canonical rna-recognition motif  domain and one or more cchc zinc knuckle motifs  <cit> .
orf <dig> proteins evolve much faster than rt proteins, leading to them being very poorly conserved between lineages.
that means that rt proteins from distantly related species still show strong homology, while orf <dig> proteins have virtually no similarity  <cit> .
in the work reported here, the eka family in the genome of bgh isolate dh <dig>  has been analysed with pipelines from the repet package, which allows de novo detection, classification and annotation of transposable elements  in whole genomes .
this method detects te sequences in the genome and groups them according to a putative common ancestor represented by a consensus sequence .
the method then identifies the matches between each te consensus and the genomic sequence  and reconstructs the te copies in the genome, even if they are nested and degenerated.
a te copy is a chain of matches between each te consensus and the genomic sequence, each match in the chain being a te fragment.
hence, a full-length te copy may correspond to several te fragments, which, when connected together, correspond to the full te consensus sequence  <cit> .
our results show that avrk <dig> and avra <dig> have evolved from part of the orf <dig> gene of class i line retrotransposons, which we have named kryze and satine, respectively.
the activity of the ancestors of these elements generated a high diversity of degenerate copies.
this provides a possible mechanism for the extensive proliferation of the eka family in the bgh genome.
these results imply that avrk <dig> and avra <dig> originated from the truncated orf <dig> of class i-line retrotransposons, in a recycling and neofunctionalization process in which the retrotransposon genes were recruited by the bgh genome as effectors.
the barley plant then evolved to recognize the presence of the bgh parasite through the presence of these retrotransposon-derived effector genes in the fungal genome.
RESULTS
avrk <dig> and avra <dig> are homologues of the orf <dig> of line  <dig> retrotransposons
our previous results indicated that the eka gene family was very large and related to tes.
thus, we based the analysis of the evolution of this family on identification and classification of te consensus sequences with homology to avrk <dig> and avra <dig>  a first step made use of bgt_rix_inari, a complete te consensus characterized in b. graminis f.sp.
tritici   <cit>  that had been classified as a line retrotransposon and contained a region highly similar to avra <dig>  we identified several fragments homologous to bgt_rix_inari in the genome of blumeria graminis f.sp.
hordei  isolate dh <dig> that were then used to build manually a consensus model for bgh that we called satine, which represents a line retrotransposon with two orfs .
orf <dig> is homologous to avra <dig>  and contains, in the 3’ region, a sequence coding for a cysteine-rich nb domain conserved between different tes  <cit> .
orf <dig> is homologous  to the reverse transcriptase and rnase h  of the retrotransposon cgt <dig> identified in the fungal plant parasite glomerella cingulata .fig.
<dig> consensus model satine, which represents a line retrotransposon with two orfs.
orf <dig> is homologous to avra <dig>  and contains, in the 3’ region, a sequence coding for a cysteine-rich nucleotide binding domain conserved between different tes .
orf <dig> is homologous to the reverse transcriptase and rnase h  of the retrotransposon cgt <dig> identified in the fungal plant parasite glomerella cingulata.
utr: untranslated region
we then refined the te annotation of the bgh genome using the tedenovo pipeline and several rounds of the teannot pipeline.
satine and two previously characterized bgh class i sine retro-elements   <cit>  were added to the repbase library  <cit>  used by the tedenovo pipeline to classify te consensus sequences.
we finally obtained a library of  <dig> te consensus sequences .
te annotation with blgr_reftes accounted for  <dig>  % of bgh genome.
class i line and ltr retrotransposons were the most abundant tes in bgh genome, accounting for  <dig>  % and  <dig>  % of bgh genome assembly, respectively .table  <dig> classification, number, content and genome coverage of te consensus sequences annotated in bgh genome.
in order to identify the te consensus sequences most similar to avra <dig> and avrk <dig> in the bgh_reftes library, we performed a blastx-based sequence comparison  <cit> .
we recovered  <dig> and  <dig> consensus sequences highly similar  to avra <dig> and avrk <dig> respectively and  <dig> te consensus sequences similar to both avra <dig> and avrk <dig> .
these consensus sequences contain  <dig> genomic copies and represent  <dig>  mbp  of the bgh annotated genome .
two te consensus sequences , classified by repet as line retrotransposons, span the whole satine consensus model .
these three consensus sequences , were used to annotate four, one and two satine-like full-length copies respectively, in the genome.
the plot of the location of the genome copies on their respective te consensus sequences shows that they are smoothly scattered along the whole consensus model, with no break points indicative of chimerization events .
the absence of break points indicates that avra <dig> is part of the original orf <dig> of the satine class i-line retrotransposon.table  <dig> metrics of the line consensus sequences homologous to avrk <dig> or avra10
line similar to both
fig.
<dig> genome te copies plotted on the consensus sequences satine , satine-like bgh_rix_g <dig>  and satine-like bgh_rix_g <dig> .
brown lines represent the part of the consensus sequence that aligns with each annotated copy.
copies are ordered according to their coordinates .
the black curve represents the depth of coverage along the reference sequence
any of the nine te consensus sequences similar to avrk <dig> could be considered full-length line retrotransposons containing both orf  <dig> and orf  <dig>  seven out these nine te consensus sequences matched  <dig> full-length fragments spanning the whole te consensus sequence.
in order to look for putative orf <dig> that had been overlooked during te search and annotation, we extracted the genome sequences of these  <dig> fragments and searched for line-orf <dig> domains within the 3000 bp downstream of the orf <dig>  pastec teclassifier  <cit>  identified the characteristic rt-rh domain at the expected location in one of those sequences .
thus, we found a potential genome copy of a full-length line retrotransposon with  <dig> orfs  in a full-length genome copy annotated by te consensus bgh_rix_g <dig> , extended to 3000 bp downstream.
the orf  <dig> of kryze is similar to avrk <dig> , and the extended 3000 bp containing the orf <dig> of kryze are similar  to the te consensus bgh_rix_g <dig> .fig.
<dig>
kryze potential genome copy containing an orf <dig> homologous to avrk <dig> containing a cysteine-rich nb domain  and a putative orf2p with rt  and rh  domains.
a blgr_v3_contig_ <dig> fa: <dig> .24230 bp corresponding to bgh_rix_g <dig> full-length copy with 3000 bp downstream region.
avrk <dig> alignment  and domains annotated by pastec classifier  are represented.
b blue lines represent the two te genomic copies mapped in the context of all the copies  plotted to the reference te consensus sequences bgh_rix_g <dig> and bgh_rix_g5646
characteristics of the eka family
the alignment of avrk <dig> and avra <dig> with the orf1p of the respective elements  indicates that both proteins have lost the c-terminal half that contains the conserved nb domain typical of an orf1p .
hence, candidates for other avr/effector genes may consist of truncated copies of the orf1p without the region containing the nb domain, thus spanning only the region homologous to avrk <dig> or avra <dig>  we extracted the  <dig> genomic copies homologous to avrk <dig> and avra <dig> and searched for the sequences spanning either the full length or the truncated orf1p .
we found  <dig> avr/effector candidates homologous to avrk <dig>   <dig> avr/effector candidates homologous to avra <dig>  and  <dig> avr/effector candidates equidistant from both avrak <dig> and avra <dig> .
the mean identity among truncated sequences was lower than the identity between the sequences homologous to the full-length orf1p for all three types of sequences .
also, the redundancy was eight times higher in full-length orf <dig> sequences than in truncated sequences .
hence, truncated sequences are less conserved than full-length orf <dig> sequences.table  <dig> number of sequences per group  and mean identity percentage in each group for full length orf <dig> and truncated sequences homologous to avrk <dig> or avra <dig>
similar to both
amean identity percentage in each group is based on translated full-length orf calculated by using the pairwise identity after realigning each pair of sequences and averaging it out for all sequences in the group.
phylogenetic analysis classifies the sequences from bgh isolate dh <dig> homologous to avrk <dig> or avra <dig> in two clades .
clade  <dig> contains mainly avrk <dig> homologues and a few equidistant sequences and groups them with the kryze orf1p and with avrk <dig> from isolate cc <dig>  clade  <dig> groups all the remaining sequences in five subclades.
subclade 2a <dig> contains only avra <dig> homologues and groups them with satine orf1p and with avra <dig> from isolate cc <dig>  the phylogeny indicates that stop codons between the avr-homologous sequence and the nb domain have appeared several times during the evolution of the eka family, since the truncated sequences appear in the same clades as the full length orf <dig> sequences .
we tested if truncated sequences were subject to different selection patterns compared to the full-length sequences, indicative of different functions.
we analysed the non-synonymous/synonymous substitution rate ratio  to detect positive selection  in the full length and truncated sequences homologous to avrk <dig> or avra <dig> .
we found evidence of positive selection with variable selective pressure among sites in the four groups of sequences analyzed  .
there were different positively selected sites in the avrk1-like truncated sequences than in the full-length te orf1p: sites 125p, 127s, 149i and 172h are positively selected in avrk1-like truncated sequences and sites 233 l, 294i, 298y and 316 l, situated after the nb domain, are positively selected in the sequences corresponding to full-length te orf1p .
for avra10-like sequences, the likelihood ratio test  did not identify significant positively selected sites either in the truncated sequences or in the full-length te.fig.
<dig> phylogenetic tree of full length and truncated sequences homologous to avrk <dig> or avra <dig> calculated using bayesian method.
circles on nodes represent posterior probability .
colours represent homology to either avrk <dig>  avra <dig> or both, and differentiate between sequences corresponding to full length orf <dig> or truncated sequences .
red and black dots indicate proteins expressed in haustoria or/and hyphae, respectively.
yellow dots indicate proteins putatively expressed in haustoria, with one significant peptide and a peptide just below the identity score threshold
fig.
<dig> identification of sites under positive selection of avrk <dig> homologues as identified under both m <dig>  and m <dig>  models.
a truncated sequences and b full-length sequences.
the vertical axis represents posterior probabilities for sites with different ω ratios  along the sequence.
positively selected sites  are highlighted on top of the graph.
*: p >  <dig> , **:p >  <dig>  .
w <dig>  w <dig> and w <dig> are the three ω estimated values in the m <dig> model
we searched for specific domains in orf <dig> predicted proteins in both full length and truncated sequences.
there was no similarity with any known orf <dig> protein.
there was weakly significant similarity  to retrovirus zinc finger-like domains in both full-length and truncated sequences, whilst predicted coiled coils were only found in full-length orf1ps.
eka protein identification
several predicted eka proteins were identified experimentally in infected epidermis  and in sporulating hyphae from 5– <dig> dpi infected barley plants .
most peptides contained proline residues, as they were identified from sequences with >8 % proline content and many were detected as hydroxyprolines.
the presence of proline is detrimental for mass spectrometry peptide fragmentation using collision induced dissociation  as the “proline effect” leads to a poor fragmentation pattern  <cit> .
this may explain the low mascot peptide scores, just above the identity threshold, for most detected peptides which contain proline-rich residues.
since proteins could only be identified with two or three peptides, it was not possible to discriminate well between isoforms of closely related proteins, hampering determination of the exact number of proteins in each sample .
however, it is clear that several eka proteins are differentially expressed in various fungal tissues, since most protein hits were identified only in either the haustoria or the hyphae , with only four proteins identified in both types of sample .
the possible  <dig> validated proteins were identified from  <dig> detected peptides in the haustoria samples and  <dig> peptides in the hyphae samples.
there was almost no overlap of identified peptides between tissues; only the peptide aaaplplr was identified in both the haustorial and hyphal samples .
most of the identified eka proteins were avra10-like, notably those in haustorial samples , with most of the hits in subclade 2a <dig> that contains satine orf1p , including a copy of the te consensus bgh_rix_g <dig> that spans the whole satine consensus model.
a copy of consensus bgh_rix_g <dig>  which also spans the whole satine consensus model, was found in a hyphal sample.
avrk1-like proteins were only found in hyphal samples .
thirteen of the  <dig> identified proteins are truncated orf1s and are thus putative avr/effectors.
most of them  are avra10-like and appear in haustorial samples, whereas only one truncated protein is avrk1-like .
additional avra- <dig> proteins in the subclade 2a <dig> were identified in haustoria with one significant peptide and a peptide just below the identity score threshold , reinforcing the hypotheses that some haustorium-specific avra <dig> effectors are expressed as proteins.table  <dig> proteomic analysis of haustoria and hyphae samples for experimental identification of eka proteins.
apeptide ddriflr is just below the identity score threshold.
discussion
the genomes of powdery mildew fungi are amongst the largest of ascomycete fungi, due to an extraordinary proliferation of tes  <cit> .
this is probably, in part, the result of the absence of the rip  pathway to control genetic parasites that is otherwise conserved in all related ascomycetes  <cit> .
te proliferation and genome expansion may have had deleterious effects on powdery mildews, entailing a considerable loss of genes that has resulted in these pathogens being entirely dependent on living host cells  <cit> .
however, te activity has also benefited powdery mildews and other eukaryotic pathogens, favoring the expansion and diversification of a broad repertoire of effector genes, as previously shown .
here, we show an additional way in which transposition activity contributed to the evolution of b. graminis, by the recycling and neofunctionalization of degenerate te products which may generate new effector genes.
the products of at least two of these genes, avrk <dig> and avra <dig> are recognized by the host plant as avirulence proteins and it is conceivable that other members of the eka family may encode avirulence genes.
our results indicate that the origin of avrk <dig> and avra <dig> is the truncated orf <dig> of the retrotransposons satine and kryze respectively .
satine and kryze are members of the i superfamily of lines, containing protein domains typical of that superfamily and showing characteristic sequence organization.
the i superfamily is the only group of lines that contains a rnaseh domain in their orf <dig>  together with the randl group and some members of the l <dig> superfamily.
additionally, the i superfamily is characterized by having an orf <dig> upstream of the orf <dig>  with zinc finger-like domains  <cit> .
our structure analysis showed that the orf <dig> of satine and kryze contains a zinc finger-like domain characteristic of orf <dig> of the line i elements.
because orf <dig> proteins are known to evolve very rapidly, it is not surprising that no homologues of satine and kryze elements were found outside the powdery mildew fungi  <cit> .
the expansion of satine and kryze lineages may have contributed to the expansion of other protein effectors of the eka family.
avrk <dig> and avra <dig> were isolated from bgh isolate cc <dig>  which is avirulent to barley plants carrying resistance genes mla <dig> and mlk <dig>  <cit> .
isolate dh <dig> is virulent to barley plants carrying mla <dig> and mlk <dig> and does not contain functional variants of avrk <dig> or avra <dig> in the corresponding loci delimited by genetic and physical mapping  <cit> .
however, this and previous works have found eka homologues in dh <dig> and other bgh isolates, many of which are transcribed .
thus, these homologues may function as effectors in these other isolates and also be avr genes recognized by r genes other than mla <dig> and mlk <dig>  we have found  <dig> truncated copies of satine and kryze orf1ps that may be avr/effector candidates because they span the region homologous to avrk <dig> or avra <dig> and lack the downstream sequence containing the nb domain .
the lower degree of conservation of truncated sequences compared to full-length orf <dig> sequences  could be due to diversifying selection concomitant with the expansive generation of effectors.
different positively selected sites were found in the avrk1-like truncated sequences than in the full-length te orf1p , which suggests that the two groups of sequences have different functions.
however, the likelihood ratio test did not find significant positively selected sites in avra <dig> homologues.
this could be due to a lower number of analyzed avra <dig> homologues and/or to the fact that avra <dig> homologues are phylogenetically very heterogeneous, in contrast to avrk <dig> homologues, which are mostly grouped in a single clade that also contains the kryze orf <dig> .
a previous analysis did not find avrk <dig> homologues in b. graminis parasitic on oats or ryegrass , while avra <dig> homologues are present in the genomes of forms of b. graminis parasitic on oats, ryegrass, barley and the closely related species rye, wheat and elymus repens  <cit> .
this implies that the eka family proliferated during the evolution of different forms of b. graminis, but the avrk <dig> clade  only proliferated after the divergence of b. graminis on oat and ryegrass from the other forms  <cit> .
hence, different eka lineages could be related to the adaptation of b. graminis to different hosts.
our results suggest that there has been a continuous process of recruitment and neofunctionalization of truncated orf <dig> copies.
diversification and amplification of this family of effector genes most likely happened as a result of strong natural selection by the host.
loss or mutation of an avr/effector protein would have resulted in the pathogen not being recognized by the plant host but would have generated selection pressure for its replacement by other effector proteins .
the concept of neofunctionalization and co-option of te sequences by host genomes was proposed by brosius and gould  <cit> , who suggested the term “exaptation” for the phenomenon of “junk” dna sequences such as tes acquiring a novel function in the genome.
more recently, genome-scale analyses have confirmed that domesticated or exapted te-derived sequences have contributed diverse and abundant regulatory and protein-coding sequences to host genomes  <cit> .
the new functions which arose from recruitment of te sequences range from their capture as cis-regulatory sequences at promoter and enhancer regions  <cit>  to the most extensive gene domestication in which single copy genes are well conserved among lineages  <cit> .
examples of te domestication are the element of the transib superfamily from which the vj system at the base of the vertebrate immune system evolved  <cit> , the herv-derived syncitin genes that play crucial roles in placenta formation  <cit>  and the non-ltr te origin of telomeres and telomerases  <cit> .
the evolution of the eka family of effectors in a plant pathogenic fungus further indicates the extraordinary diversity of functions which have evolved from retroelements.fig.
<dig> model for the evolution of the eka family by co-option as effectors by bgh genome.
<dig>  genome rearrangements after retrotransposon activity produce degenerate copies of line retrotransposons in the bgh genome.
some of these copies are truncated orf1s.
<dig>  one truncated orf <dig> is co-opted as effector e <dig> that enhances pathogenicity.
<dig>  plant host evolves a resistance gene r <dig> that recognizes the presence of e <dig>   <dig>  e <dig> becomes an avirulence gene  if it is recognized by r <dig> in a gene-for-gene interaction.
<dig>  avr <dig> mutates to avoid recognition by r <dig>   <dig>  bgh co-opts other degenerate copies as effectors that contribute to enhanced pathogenicity if they are not recognized by host r genes.
during this process, bgh genome and eka copies are evolving, and the copy co-opted at point  <dig> may not have existed at point  <dig> and could have come from a different eka ancestor
avrk <dig> and avra <dig> have been shown to function as effectors because they enhance bgh infectivity  <cit> .
it is predicted that avr genes recognized by plant r genes should confer a selective advantage if they are to be retained in the pathogen population .
while avrk <dig> and avra <dig> have effector activities as well as being recognized as avirulence proteins , their precise functions in the interaction of bgh with its host are unknown.
there are several examples that show the transcription of avrk <dig> and avra <dig> and other members of the eka family .
here, we provide the first experimental evidence indicating the production of eka proteins by bgh.
we identified  <dig> proteins putatively expressed in the haustorial samples and  <dig> proteins putatively expressed in the hyphal samples .
this makes a ratio of 1: <dig> for proteins identified in the haustoria versus the hyphae that, despite the difficulty to determine the exact number of proteins actually expressed, is clearly higher than the ratio found in a global proteomic analysis of these tissues, which is 1: <dig>   <cit> .
also,  <dig> and  <dig> different peptide sequences were identified in haustorial and hyphal samples respectively.
together, these data indicate that the eka proteins may be more abundant and diverse in haustoria than in sporulating hyphae.
this is consistent with the eka proteins accumulating or being secreted in association with haustorial structures like other fungal effectors  <cit> , although the secretion mechanism is unknown.
the identified peptides associated with eka proteins are almost exclusive to either haustoria or hyphae, with only one peptide shared by both types of samples , implying that different eka proteins may have specific functions in different fungal tissues.
these functions may also be specific to the eka protein subfamily, since most of the haustorium-associated proteins are avra10-like but none of the avrk1-like proteins were found in haustoria.
most of the avra10-like haustorium-associated proteins were truncated, unlike avrk1-like proteins, which were mainly full length and were thus probably functional line orf1s.
the functions of the eka proteins could be related to their biochemical or structural properties.
on the biochemical side, they are proline-rich.
one example of a fungal proline-rich protein  is expressed exclusively in plants and has a role in biotrophy during colletotrichum-host interactions  <cit> .
this gene has orthologs in c. higginsianum and passalora fulva  that are also proline-rich and are involved in chitin sequestration and camouflage to avoid plant recognition  <cit> .
on the structural side, the orf1p of superfamily i of line tes, from which the eka family derives, contains non-canonical rrm domains through its whole length  <cit> .
avrk <dig> and avra <dig> and other eka members may have conserved part of those domains.
both truncated and full length orf <dig> predicted proteins show weak similarities with zinc finger-like domains typical of retroviral gag-proteins.
these domains are involved in protein interactions with rna, dna or other proteins  <cit> .
any of the properties of these domains could have been retained to give a novel function to avrk <dig>  avra <dig> and other related avr/effector candidates and thus a selective advantage to bgh individuals carrying them.
functional innovation by exaptation has been related to extremely stressful situations or crises such as the mass extinction  <dig> mya that gave rise to mammals  <cit> .
there are several examples of increased te activity associated with stress in insects and plants  <cit> .
indeed, te mediated genetic variation can be fundamental in the host genome’s evolutionary response to stress, facilitating the adaptation of populations and species to changing environments  <cit> .
the role of tes in parasite-host coevolution as mutators which increase parasite genomic plasticity has been discussed  <cit> .
recently, retrotransposons have been shown to increase pathogenesis and virulence by silencing either pathogen avirulence genes or plant defense mechanisms  <cit> .
here, we provide evidence of a direct role of retrotransposons in virulence as dynamic reservoirs from which new effector genes evolve.
it is especially striking that the host plant recognizes the presence of the powdery mildew parasite by the presence of genes which evolved from a genomic parasite – a retrotransposon family – of the fungus.
CONCLUSIONS
transposable elements account for 67 % of bgh genome size, and class i-line retrotransposons are the most abundant in the bgh genome, representing  <dig>  % of genome coverage and  <dig>  % of the te content .
the sequences homologous to avrk <dig> and avra <dig>  within the repetitive dna landscape of the bgh genome have been identified and analysed, and the origin of these avr/effector genes has been determined to be the truncated orf <dig> of the line retrotransposons satine and kryze .
thus, avrk <dig> and avra <dig> genes have been co-opted in the bgh genome as effectors from truncated copies of retrotransposons.
dna repeats of satine-like and kryze-like lineages represent at least  <dig>  % of the genome assembly .
these data indicate a long-standing activity of these two elements in bgh genome, during which a high diversity of degenerate copies has been generated.
new effector genes may have evolved from these degenerated copies in a recycling and neofunctionalization process driven by the coevolution between bgh and its host plant .
we have found  <dig> truncated copies of satine and kryze orf1s that may be avr/effector candidates  and peptides that indicate translation of these genes .
these candidates have arisen several times during the evolution of the eka family , and belong to different lineages that could be related with the adaptation of b. graminis to different hosts.

 plant pathogens secrete effectors to influence host metabolism or defense mechanisms to provide an environment for successful infection. effectors can be recognized by plant resistance genes, eliciting effector-triggered immunity in the plant to prevent further infection. effectors that are recognized by the plant are known as avirulence proteins in the context of gene-for-gene interactions. alteration of avr genes enables parasites to avoid r-dependent recognition and thus overcome host resistance. the avrk and avra genes of the barley powdery mildew fungus, blumeria graminis f.sp. hordei, encode proteins which have a dual role as effectors and as avr proteins, the presence of which is recognized by the mlk and mla r-proteins. these genes were cloned from isolate cc by genetic and physical mapping. numerous lines of genetic and biological evidence indicate their dual function. the avr genes each co-segregate with the respective avirulence phenotype, have a functional open reading frame in avirulent but not virulent isolates, and are expressed by isolates avirulent to the corresponding r-gene. the specific recognition of avrk and avra by mlk and mla r-proteins and subsequent cell death was shown in two independent series of experiments by transient expression of avrk and avra in barley plants carrying mlk or mla resistance genes. specific recognition of avrk and avra also induced inaccessibility to subsequent infection and reduced fungal sporulation. their effector function was demonstrated in two independent experiments. first, when they were transiently overexpressed in susceptible plants lacking the corresponding r-gene, they increased the infectivity of bgh. second, host-induced gene silencing of these genes in susceptible plants caused a reduction in haustorium formation by bgh. further evidence for effector and avr functions has been obtained for avra this gene is expressed at low levels in conidia that have just landed on the leaf surface, followed by rapid induction within 6 h and high expression until 24 h after inoculation, with a drop thereafter. the interaction between the mla protein and the wrky transcription factor depended on the presence of avra protein for defense gene activation. an ems-induced point mutation of avra producing premature termination of the coding sequence, rendered the pathogen virulent to plants carrying the mla resistance gene. natural bgh isolates virulent to mla or mlk do not have functional variants of avrk or avra in the corresponding loci, in many cases due to the fusion of the avr genes with retrotransposon sequences. avrk and avra are homologues, and are the first discovered members of the eka gene family, with more than homologues in the bgh genome. the eka family has no known homologues outside powdery mildew fungi but is present in different formae speciales of blumeria graminis and in powdery mildew species infecting different host such as pea, grape, plantain or arabidopsis thaliana. a previous analysis showed that these avrk and avra homologues are frequently found in the same orf as the nucleotide binding domain of a line retrotransposon, that the avr-homologous and nb domains are expressed in different bgh isolates as a single transcript, and that both type of sequences have coevolved. line retrotransposons are almost ubiquitous in fungi, plants and animals. they are able to replicate autonomously, and their mobility is dependent on target-primed reverse transcription. these elements typically consist of an orf containing a gene encoding a reverse transcriptase and an endonuclease. in addition, orf usually found in the l i and jockey groups, may have been acquired independently on multiple occasions during the evolution of line elements. the best studied orf protein is that in the l superfamily of line elements. orf1p is thought to assist l retrovirus-like particles to gain access to the nucleus, where it can interact with genomic dna and thus initiate integration through target primed reverse transcription. the orf1p of the second major superfamily of lines is only poorly characterized and its function is not yet fully understood. it usually contains at least one non-canonical rna-recognition motif domain and one or more cchc zinc knuckle motifs. orf proteins evolve much faster than rt proteins, leading to them being very poorly conserved between lineages. that means that rt proteins from distantly related species still show strong homology, while orf proteins have virtually no similarity. in the work reported here, the eka family in the genome of bgh isolate dh has been analysed with pipelines from the repet package, which allows de novo detection, classification and annotation of transposable elements in whole genomes. this method detects te sequences in the genome and groups them according to a putative common ancestor represented by a consensus sequence. the method then identifies the matches between each te consensus and the genomic sequence and reconstructs the te copies in the genome, even if they are nested and degenerated. a te copy is a chain of matches between each te consensus and the genomic sequence, each match in the chain being a te fragment. hence, a full-length te copy may correspond to several te fragments, which, when connected together, correspond to the full te consensus sequence. our results show that avrk and avra have evolved from part of the orf gene of class i line retrotransposons, which we have named kryze and satine, respectively. the activity of the ancestors of these elements generated a high diversity of degenerate copies. this provides a possible mechanism for the extensive proliferation of the eka family in the bgh genome. these results imply that avrk and avra originated from the truncated orf of class i-line retrotransposons, in a recycling and neofunctionalization process in which the retrotransposon genes were recruited by the bgh genome as effectors. the barley plant then evolved to recognize the presence of the bgh parasite through the presence of these retrotransposon-derived effector genes in the fungal genome.  avrk and avra are homologues of the orf of line retrotransposons our previous results indicated that the eka gene family was very large and related to tes. thus, we based the analysis of the evolution of this family on identification and classification of te consensus sequences with homology to avrk and avra a first step made use of bgt_rix_inari, a complete te consensus characterized in b. graminis f.sp. tritici that had been classified as a line retrotransposon and contained a region highly similar to avra we identified several fragments homologous to bgt_rix_inari in the genome of blumeria graminis f.sp. hordei isolate dh that were then used to build manually a consensus model for bgh that we called satine, which represents a line retrotransposon with two orfs. orf is homologous to avra and contains, in the 3’ region, a sequence coding for a cysteine-rich nb domain conserved between different tes. orf is homologous to the reverse transcriptase and rnase h of the retrotransposon cgt identified in the fungal plant parasite glomerella cingulata.fig. consensus model satine, which represents a line retrotransposon with two orfs. orf is homologous to avra and contains, in the 3’ region, a sequence coding for a cysteine-rich nucleotide binding domain conserved between different tes. orf is homologous to the reverse transcriptase and rnase h of the retrotransposon cgt identified in the fungal plant parasite glomerella cingulata. utr: untranslated region we then refined the te annotation of the bgh genome using the tedenovo pipeline and several rounds of the teannot pipeline. satine and two previously characterized bgh class i sine retro-elements were added to the repbase library used by the tedenovo pipeline to classify te consensus sequences. we finally obtained a library of te consensus sequences. te annotation with blgr_reftes accounted for  % of bgh genome. class i line and ltr retrotransposons were the most abundant tes in bgh genome, accounting for  % and  % of bgh genome assembly, respectively.table classification, number, content and genome coverage of te consensus sequences annotated in bgh genome. in order to identify the te consensus sequences most similar to avra and avrk in the bgh_reftes library, we performed a blastx-based sequence comparison. we recovered and consensus sequences highly similar to avra and avrk respectively and te consensus sequences similar to both avra and avrk. these consensus sequences contain genomic copies and represent mbp of the bgh annotated genome. two te consensus sequences, classified by repet as line retrotransposons, span the whole satine consensus model. these three consensus sequences, were used to annotate four, one and two satine-like full-length copies respectively, in the genome. the plot of the location of the genome copies on their respective te consensus sequences shows that they are smoothly scattered along the whole consensus model, with no break points indicative of chimerization events. the absence of break points indicates that avra is part of the original orf of the satine class i-line retrotransposon.table metrics of the line consensus sequences homologous to avrk or avra10 line similar to both fig. genome te copies plotted on the consensus sequences satine, satine-like bgh_rix_g and satine-like bgh_rix_g. brown lines represent the part of the consensus sequence that aligns with each annotated copy. copies are ordered according to their coordinates. the black curve represents the depth of coverage along the reference sequence any of the nine te consensus sequences similar to avrk could be considered full-length line retrotransposons containing both orf and orf seven out these nine te consensus sequences matched full-length fragments spanning the whole te consensus sequence. in order to look for putative orf that had been overlooked during te search and annotation, we extracted the genome sequences of these fragments and searched for line-orf domains within the 3000 bp downstream of the orf pastec teclassifier identified the characteristic rt-rh domain at the expected location in one of those sequences. thus, we found a potential genome copy of a full-length line retrotransposon with orfs in a full-length genome copy annotated by te consensus bgh_rix_g, extended to 3000 bp downstream. the orf of kryze is similar to avrk, and the extended 3000 bp containing the orf of kryze are similar to the te consensus bgh_rix_g.fig. kryze potential genome copy containing an orf homologous to avrk containing a cysteine-rich nb domain and a putative orf2p with rt and rh domains. a blgr_v3_contig_ fa:.24230 bp corresponding to bgh_rix_g full-length copy with 3000 bp downstream region. avrk alignment and domains annotated by pastec classifier are represented. b blue lines represent the two te genomic copies mapped in the context of all the copies plotted to the reference te consensus sequences bgh_rix_g and bgh_rix_g5646 characteristics of the eka family the alignment of avrk and avra with the orf1p of the respective elements indicates that both proteins have lost the c-terminal half that contains the conserved nb domain typical of an orf1p. hence, candidates for other avr/effector genes may consist of truncated copies of the orf1p without the region containing the nb domain, thus spanning only the region homologous to avrk or avra we extracted the genomic copies homologous to avrk and avra and searched for the sequences spanning either the full length or the truncated orf1p. we found avr/effector candidates homologous to avrk avr/effector candidates homologous to avra and avr/effector candidates equidistant from both avrak and avra. the mean identity among truncated sequences was lower than the identity between the sequences homologous to the full-length orf1p for all three types of sequences. also, the redundancy was eight times higher in full-length orf sequences than in truncated sequences. hence, truncated sequences are less conserved than full-length orf sequences.table number of sequences per group and mean identity percentage in each group for full length orf and truncated sequences homologous to avrk or avra similar to both amean identity percentage in each group is based on translated full-length orf calculated by using the pairwise identity after realigning each pair of sequences and averaging it out for all sequences in the group. phylogenetic analysis classifies the sequences from bgh isolate dh homologous to avrk or avra in two clades. clade contains mainly avrk homologues and a few equidistant sequences and groups them with the kryze orf1p and with avrk from isolate cc clade groups all the remaining sequences in five subclades. subclade 2a contains only avra homologues and groups them with satine orf1p and with avra from isolate cc the phylogeny indicates that stop codons between the avr-homologous sequence and the nb domain have appeared several times during the evolution of the eka family, since the truncated sequences appear in the same clades as the full length orf sequences. we tested if truncated sequences were subject to different selection patterns compared to the full-length sequences, indicative of different functions. we analysed the non-synonymous/synonymous substitution rate ratio to detect positive selection in the full length and truncated sequences homologous to avrk or avra. we found evidence of positive selection with variable selective pressure among sites in the four groups of sequences analyzed. there were different positively selected sites in the avrk1-like truncated sequences than in the full-length te orf1p: sites 125p, 127s, 149i and 172h are positively selected in avrk1-like truncated sequences and sites 233 l, 294i, 298y and 316 l, situated after the nb domain, are positively selected in the sequences corresponding to full-length te orf1p. for avra10-like sequences, the likelihood ratio test did not identify significant positively selected sites either in the truncated sequences or in the full-length te.fig. phylogenetic tree of full length and truncated sequences homologous to avrk or avra calculated using bayesian method. circles on nodes represent posterior probability. colours represent homology to either avrk avra or both, and differentiate between sequences corresponding to full length orf or truncated sequences. red and black dots indicate proteins expressed in haustoria or/and hyphae, respectively. yellow dots indicate proteins putatively expressed in haustoria, with one significant peptide and a peptide just below the identity score threshold fig. identification of sites under positive selection of avrk homologues as identified under both m and m models. a truncated sequences and b full-length sequences. the vertical axis represents posterior probabilities for sites with different ω ratios along the sequence. positively selected sites are highlighted on top of the graph. *: p > , **:p > . w w and w are the three ω estimated values in the m model we searched for specific domains in orf predicted proteins in both full length and truncated sequences. there was no similarity with any known orf protein. there was weakly significant similarity to retrovirus zinc finger-like domains in both full-length and truncated sequences, whilst predicted coiled coils were only found in full-length orf1ps. eka protein identification several predicted eka proteins were identified experimentally in infected epidermis and in sporulating hyphae from 5– dpi infected barley plants. most peptides contained proline residues, as they were identified from sequences with >8 % proline content and many were detected as hydroxyprolines. the presence of proline is detrimental for mass spectrometry peptide fragmentation using collision induced dissociation as the “proline effect” leads to a poor fragmentation pattern. this may explain the low mascot peptide scores, just above the identity threshold, for most detected peptides which contain proline-rich residues. since proteins could only be identified with two or three peptides, it was not possible to discriminate well between isoforms of closely related proteins, hampering determination of the exact number of proteins in each sample. however, it is clear that several eka proteins are differentially expressed in various fungal tissues, since most protein hits were identified only in either the haustoria or the hyphae, with only four proteins identified in both types of sample. the possible validated proteins were identified from detected peptides in the haustoria samples and peptides in the hyphae samples. there was almost no overlap of identified peptides between tissues; only the peptide aaaplplr was identified in both the haustorial and hyphal samples. most of the identified eka proteins were avra10-like, notably those in haustorial samples, with most of the hits in subclade 2a that contains satine orf1p, including a copy of the te consensus bgh_rix_g that spans the whole satine consensus model. a copy of consensus bgh_rix_g which also spans the whole satine consensus model, was found in a hyphal sample. avrk1-like proteins were only found in hyphal samples. thirteen of the identified proteins are truncated orf1s and are thus putative avr/effectors. most of them are avra10-like and appear in haustorial samples, whereas only one truncated protein is avrk1-like. additional avra- proteins in the subclade 2a were identified in haustoria with one significant peptide and a peptide just below the identity score threshold, reinforcing the hypotheses that some haustorium-specific avra effectors are expressed as proteins.table proteomic analysis of haustoria and hyphae samples for experimental identification of eka proteins. apeptide ddriflr is just below the identity score threshold. discussion the genomes of powdery mildew fungi are amongst the largest of ascomycete fungi, due to an extraordinary proliferation of tes. this is probably, in part, the result of the absence of the rip pathway to control genetic parasites that is otherwise conserved in all related ascomycetes. te proliferation and genome expansion may have had deleterious effects on powdery mildews, entailing a considerable loss of genes that has resulted in these pathogens being entirely dependent on living host cells. however, te activity has also benefited powdery mildews and other eukaryotic pathogens, favoring the expansion and diversification of a broad repertoire of effector genes, as previously shown. here, we show an additional way in which transposition activity contributed to the evolution of b. graminis, by the recycling and neofunctionalization of degenerate te products which may generate new effector genes. the products of at least two of these genes, avrk and avra are recognized by the host plant as avirulence proteins and it is conceivable that other members of the eka family may encode avirulence genes. our results indicate that the origin of avrk and avra is the truncated orf of the retrotransposons satine and kryze respectively. satine and kryze are members of the i superfamily of lines, containing protein domains typical of that superfamily and showing characteristic sequence organization. the i superfamily is the only group of lines that contains a rnaseh domain in their orf together with the randl group and some members of the l superfamily. additionally, the i superfamily is characterized by having an orf upstream of the orf with zinc finger-like domains. our structure analysis showed that the orf of satine and kryze contains a zinc finger-like domain characteristic of orf of the line i elements. because orf proteins are known to evolve very rapidly, it is not surprising that no homologues of satine and kryze elements were found outside the powdery mildew fungi. the expansion of satine and kryze lineages may have contributed to the expansion of other protein effectors of the eka family. avrk and avra were isolated from bgh isolate cc which is avirulent to barley plants carrying resistance genes mla and mlk. isolate dh is virulent to barley plants carrying mla and mlk and does not contain functional variants of avrk or avra in the corresponding loci delimited by genetic and physical mapping. however, this and previous works have found eka homologues in dh and other bgh isolates, many of which are transcribed. thus, these homologues may function as effectors in these other isolates and also be avr genes recognized by r genes other than mla and mlk we have found truncated copies of satine and kryze orf1ps that may be avr/effector candidates because they span the region homologous to avrk or avra and lack the downstream sequence containing the nb domain. the lower degree of conservation of truncated sequences compared to full-length orf sequences could be due to diversifying selection concomitant with the expansive generation of effectors. different positively selected sites were found in the avrk1-like truncated sequences than in the full-length te orf1p, which suggests that the two groups of sequences have different functions. however, the likelihood ratio test did not find significant positively selected sites in avra homologues. this could be due to a lower number of analyzed avra homologues and/or to the fact that avra homologues are phylogenetically very heterogeneous, in contrast to avrk homologues, which are mostly grouped in a single clade that also contains the kryze orf. a previous analysis did not find avrk homologues in b. graminis parasitic on oats or ryegrass, while avra homologues are present in the genomes of forms of b. graminis parasitic on oats, ryegrass, barley and the closely related species rye, wheat and elymus repens. this implies that the eka family proliferated during the evolution of different forms of b. graminis, but the avrk clade only proliferated after the divergence of b. graminis on oat and ryegrass from the other forms. hence, different eka lineages could be related to the adaptation of b. graminis to different hosts. our results suggest that there has been a continuous process of recruitment and neofunctionalization of truncated orf copies. diversification and amplification of this family of effector genes most likely happened as a result of strong natural selection by the host. loss or mutation of an avr/effector protein would have resulted in the pathogen not being recognized by the plant host but would have generated selection pressure for its replacement by other effector proteins. the concept of neofunctionalization and co-option of te sequences by host genomes was proposed by brosius and gould, who suggested the term “exaptation” for the phenomenon of “junk” dna sequences such as tes acquiring a novel function in the genome. more recently, genome-scale analyses have confirmed that domesticated or exapted te-derived sequences have contributed diverse and abundant regulatory and protein-coding sequences to host genomes. the new functions which arose from recruitment of te sequences range from their capture as cis-regulatory sequences at promoter and enhancer regions to the most extensive gene domestication in which single copy genes are well conserved among lineages. examples of te domestication are the element of the transib superfamily from which the vj system at the base of the vertebrate immune system evolved, the herv-derived syncitin genes that play crucial roles in placenta formation and the non-ltr te origin of telomeres and telomerases. the evolution of the eka family of effectors in a plant pathogenic fungus further indicates the extraordinary diversity of functions which have evolved from retroelements.fig. model for the evolution of the eka family by co-option as effectors by bgh genome. genome rearrangements after retrotransposon activity produce degenerate copies of line retrotransposons in the bgh genome. some of these copies are truncated orf1s. one truncated orf is co-opted as effector e that enhances pathogenicity. plant host evolves a resistance gene r that recognizes the presence of e e becomes an avirulence gene if it is recognized by r in a gene-for-gene interaction. avr mutates to avoid recognition by r bgh co-opts other degenerate copies as effectors that contribute to enhanced pathogenicity if they are not recognized by host r genes. during this process, bgh genome and eka copies are evolving, and the copy co-opted at point may not have existed at point and could have come from a different eka ancestor avrk and avra have been shown to function as effectors because they enhance bgh infectivity. it is predicted that avr genes recognized by plant r genes should confer a selective advantage if they are to be retained in the pathogen population. while avrk and avra have effector activities as well as being recognized as avirulence proteins, their precise functions in the interaction of bgh with its host are unknown. there are several examples that show the transcription of avrk and avra and other members of the eka family. here, we provide the first experimental evidence indicating the production of eka proteins by bgh. we identified proteins putatively expressed in the haustorial samples and proteins putatively expressed in the hyphal samples. this makes a ratio of 1: for proteins identified in the haustoria versus the hyphae that, despite the difficulty to determine the exact number of proteins actually expressed, is clearly higher than the ratio found in a global proteomic analysis of these tissues, which is 1:. also, and different peptide sequences were identified in haustorial and hyphal samples respectively. together, these data indicate that the eka proteins may be more abundant and diverse in haustoria than in sporulating hyphae. this is consistent with the eka proteins accumulating or being secreted in association with haustorial structures like other fungal effectors, although the secretion mechanism is unknown. the identified peptides associated with eka proteins are almost exclusive to either haustoria or hyphae, with only one peptide shared by both types of samples, implying that different eka proteins may have specific functions in different fungal tissues. these functions may also be specific to the eka protein subfamily, since most of the haustorium-associated proteins are avra10-like but none of the avrk1-like proteins were found in haustoria. most of the avra10-like haustorium-associated proteins were truncated, unlike avrk1-like proteins, which were mainly full length and were thus probably functional line orf1s. the functions of the eka proteins could be related to their biochemical or structural properties. on the biochemical side, they are proline-rich. one example of a fungal proline-rich protein is expressed exclusively in plants and has a role in biotrophy during colletotrichum-host interactions. this gene has orthologs in c. higginsianum and passalora fulva that are also proline-rich and are involved in chitin sequestration and camouflage to avoid plant recognition. on the structural side, the orf1p of superfamily i of line tes, from which the eka family derives, contains non-canonical rrm domains through its whole length. avrk and avra and other eka members may have conserved part of those domains. both truncated and full length orf predicted proteins show weak similarities with zinc finger-like domains typical of retroviral gag-proteins. these domains are involved in protein interactions with rna, dna or other proteins. any of the properties of these domains could have been retained to give a novel function to avrk avra and other related avr/effector candidates and thus a selective advantage to bgh individuals carrying them. functional innovation by exaptation has been related to extremely stressful situations or crises such as the mass extinction mya that gave rise to mammals. there are several examples of increased te activity associated with stress in insects and plants. indeed, te mediated genetic variation can be fundamental in the host genome’s evolutionary response to stress, facilitating the adaptation of populations and species to changing environments. the role of tes in parasite-host coevolution as mutators which increase parasite genomic plasticity has been discussed. recently, retrotransposons have been shown to increase pathogenesis and virulence by silencing either pathogen avirulence genes or plant defense mechanisms. here, we provide evidence of a direct role of retrotransposons in virulence as dynamic reservoirs from which new effector genes evolve. it is especially striking that the host plant recognizes the presence of the powdery mildew parasite by the presence of genes which evolved from a genomic parasite – a retrotransposon family – of the fungus.  transposable elements account for 67 % of bgh genome size, and class i-line retrotransposons are the most abundant in the bgh genome, representing  % of genome coverage and  % of the te content. the sequences homologous to avrk and avra within the repetitive dna landscape of the bgh genome have been identified and analysed, and the origin of these avr/effector genes has been determined to be the truncated orf of the line retrotransposons satine and kryze. thus, avrk and avra genes have been co-opted in the bgh genome as effectors from truncated copies of retrotransposons. dna repeats of satine-like and kryze-like lineages represent at least  % of the genome assembly. these data indicate a long-standing activity of these two elements in bgh genome, during which a high diversity of degenerate copies has been generated. new effector genes may have evolved from these degenerated copies in a recycling and neofunctionalization process driven by the coevolution between bgh and its host plant. we have found truncated copies of satine and kryze orf1s that may be avr/effector candidates and peptides that indicate translation of these genes. these candidates have arisen several times during the evolution of the eka family, and belong to different lineages that could be related with the adaptation of b. graminis to different hosts. 
we tested the hypothesis that the eka family originated from degenerate copies of class i line retrotransposons by analysing the eka family in the genome of bgh isolate dh with bioinformatic tools specially developed for the analysis of transposable elements in genomes. our analyses indicate that avrk and avra have evolved from part of the orf gene of class i line retrotransposons. the co-option of avra and avrk as effectors from truncated copies of retrotransposons explains the huge number of homologues in bgh genome that could act as dynamic reservoirs from which new effector genes may evolve. we also experimentally identified peptides that indicated the translation of several predicted proteins from eka family members, which had higher relative abundance in haustoria than in hyphae. avrk and avra have more than homologues in bgh genome, forming the eka gene family.
 the avrk and avra avirulence genes encode effectors that increase the pathogenicity of the fungus blumeria graminis f.sp. hordei, the powdery mildew pathogen, in susceptible barley plants. in resistant barley, mlk and mla resistance proteins recognize the presence of avrk and avra eliciting the hypersensitive response typical of gene for gene interactions. avrk and avra have more than homologues in bgh genome, forming the eka gene family.  we tested the hypothesis that the eka family originated from degenerate copies of class i line retrotransposons by analysing the eka family in the genome of bgh isolate dh with bioinformatic tools specially developed for the analysis of transposable elements in genomes. the class i line retrotransposon copies homologous to avrk and avra represent  % of the bgh annotated genome and, among them, we identified avr/effector candidate genes. we also experimentally identified peptides that indicated the translation of several predicted proteins from eka family members, which had higher relative abundance in haustoria than in hyphae.  our analyses indicate that avrk and avra have evolved from part of the orf gene of class i line retrotransposons. the co-option of avra and avrk as effectors from truncated copies of retrotransposons explains the huge number of homologues in bgh genome that could act as dynamic reservoirs from which new effector genes may evolve. these data provide further evidence for recruitment of retrotransposons in the evolution of new biological functions. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords barleypowdery mildewblumeria graminiseffectorretrotransposonlineco-optionissue-copyright-statement© the author 2015 