BACKGROUND
present in the environment as free-living organisms or in symbiosis with other organisms, fungi have a major impact on human society.
besides being major contributors to the decomposition and recycling of nutrients, they are largely employed in food production, biotechnologies and agricultural systems.
however, these microorganisms also cause devastating diseases, particularly of plants, representing a major threat to global food security.
it is noteworthy that a significant number of plant pathogenic fungi are able to cause serious human and animal diseases, for which there are very few effective therapeutic agents.
verticillium dahliae  and v. albo-atrum   cause very disruptive vascular diseases in over  <dig> plant species, including vegetable, ornamental and tree crops  <cit> .
these microorganisms penetrate the root system of their hosts and induce typical leaf wilt symptoms by spreading throughout the xylem vessels and disrupting water transport.
compared to va, vd is generally characterized by a broader host range and geographical distribution, and the capability to produce longer lasting soil resting structures called microsclerotia.
no sexual reproduction has been reported in either vd or va, thus implicating somatic mutation as a driving force in their evolution.
comparative analysis of the recently sequenced genomes of the isolates vdls.
<dig>  and vams.
<dig> , showed that they are 97% identical and that vdls.
<dig> contains regions in chromosomes three and four that are not present in the va isolate  <cit> .
these lineage-specific  regions, which encompass about  <dig> % of the fungal genome, comprise about  <dig> protein-coding genes and are also enriched in repetitive sequences corresponding to both previously known  <cit>  and as-yet uncharacterized transposon-like sequences  <cit> .
transposable elements  are found in virtually every prokaryotic and eukaryotic genome where they can exert a multifaceted mutagenic activity that leads to changes in chromosome architecture, generation of new regulatory networks and increases in the protein repertoire  <cit> .
on the basis of structural-functional characteristics, tes have been separated into two major classes each comprising subclasses or orders, superfamilies, families and subfamilies  <cit> .
the elements of class i, retrotransposons, replicate by a “copy-and-paste” mechanism that generates rna intermediates, which are subsequently reverse transcribed into double-stranded  dna by te-encoded enzymes.
these retroelements have been recently classified into five orders comprised of long terminal repeat  elements, dictyostelium intermediate repeat sequence , penelope-like elements , and long and short interspersed nuclear elements   <cit> .
among the most widespread retrotransposons are the ltr superfamilies gypsy and copia, which include two orfs coding for the structural virus-like protein gag and the reverse transcriptase /integrase  enzyme pol.
class ii elements, dna transposons, are mobilized via the formation of dna intermediates.
if the intermediate is dsdna, the transposition is called “conservative” and the elements are of the “cut-and-paste” type.
complete, alias autonomous, tes of this type are generally delimited by terminal inverted repeats  and encode a single transposase.
the transposase binds conserved regions within the tirs, mostly direct short tandem duplications, and catalyzes the excision of the complete element, which then reintegrates in a new genomic location.
when te mobilization proceeds via single-stranded  dna and does not involve the element excision it is defined as “replicative”, a mode of transposition that characterizes the more complex helitron- and maverick-like transposons  <cit> .
although fungal genomes generally contain fewer repetitive sequences than do higher eukaryotes  <cit> , tes have played critical roles in the evolution of their fungal hosts and, in particular, of the phytopathogens.
in the rice blast fungus, magnaporthe oryzae, for example, tes were found to cluster in regions having high recombination rates and gene duplication events  <cit> .
similarly, in the black mold aspergillus niger, specific retrotransposon-mediated recombinations have led to inversions of genomic regions  <cit> .
in addition, host-specific toxin genes of cochliobolus carbonum <cit>  and alternaria alternata <cit> , as well as host-specificity genes of m. oryzae <cit>  are situated in transposon-rich regions of the genome.
because gene products conferring host-specificity can be recognized by plant host defense receptors, it has been hypothesized that this proximity could allow expansion of host range through te-mediated gene loss  <cit> .
in addition, te distribution can be extremely variable between isolates of a single fungal species, and tes have been used as markers to distinguish genetically divergent populations, and for the identification of subpopulations  <cit> .
introgression of mobile elements into the genome is potentially deleterious, and cells have evolved different processes for their elimination, including silencing by rna interference and repeat-induced point  mutation  <cit> .
the introduction of rip mutations in the dna of fungi is linked to sexual reproduction, occurring at the time of meiosis, and the process likely arose as a genome defense mechanism against the intrusion of repetitive dna sequences.
the rip machinery recognizes duplicated sequences longer than 400 bp and sharing more than 80% identity, and introduces in both copies c:g to t:a transitions  <cit> .
noticeably, rip has been detected in the asexual fungi a. nigerpenicillium crysogenum and v. dahliae <cit> , suggesting the existence of an ancestral or cryptic sexual state in these species.
interestingly, rip mutations in vdls.
<dig> affected members of the gypsy but not of the copia superfamilies of retrotransposons, indicating either a differential susceptibility to rip by different types of tes, or introgression of already riped sequences from a sexual organism  <cit> .
to further investigate the nature and extent of genetic diversity in v. dahliae, we have undertaken an analysis of tes in this species.
we report here the identification and characterization of class i and ii transposons in v. dahliae, an analysis of their distribution in v. dahliae and v. albo-atrum, and discuss their potential role as generators of diversity in the verticillium genomes.
RESULTS
transposable elements in verticillium dahliae genome
using a combination of bioinformatics predictions and manual inspections, we identified  <dig> complete class i retrotransposons and  <dig> class ii “cut-and-paste” dna transposons in the vdls.
<dig> genome.
based on comparison to consensus sequence structures  <cit> , the retrotransposons were identified as members of the ltr class copia and gypsy superfamilies, and line superfamily i, and the dna tes as subclass i members of the tir superfamilies tc1/mariner, activator  and mutator  .
ltr retrotransposons of superfamily copia
we identified  <dig> full-length copia-like tes, referred to as vdltre  <dig> and  <dig>  respectively .
the  <dig> copies of vdltre <dig> were  <dig>  bp in length, flanked by 213 bp ltrs, and contained a single orf with fused gag and pol sequences predicted to encode a  <dig> amino acid  polyprotein.
in contrast, the two copies of vdltre <dig> had shorter  ltrs, and gag and pol orfs separated by a uga stop codon.
this rarely observed orf organization requires a leaky stop codon for translation read-through of the pol orf 3’ into the gag orf.
a conserved caryya sequence has been shown to be important for the stop codon read-through  <cit> , however a search of vdltre <dig> showed that it lacked this canonical sequence motif.
a motif search identified in both vdltre <dig> and vdltre <dig> an integrase core domain located n-terminally to a reverse transcriptase domain, placing both elements as members of the ty1/copia family of retroelements  <cit> .
these retrotransposases shared the highest sequences similarities with hypothetical proteins of the fungal animal pathogen penicillium marneffei.
finally, a genome-wide search was conducted that identified two vdltre <dig> solo-ltrs.
such sequences are commonly observed remnants of ltr retrotransposons, and likely arise from recombination between the direct repeat sequences that flank retroelements  <cit> .
ltr retrotransposons of superfamily gypsy
three families of gypsy-like elements were discovered and referred to as vdltre <dig>  vdltre <dig>  and vdltre <dig> , and corresponded to vdf <dig> and vdf <dig>  previously identified as v. dahliae retrotransposon-like sequences  <cit> .
these elements were ~  <dig>  to  <dig>  kb in size,  <dig> % identical at the nucleotide level, encoded nearly identical gag and pol proteins, and possessed ltrs of  <dig>   <dig> and 455 bp, respectively .
there were  <dig> full-length elements and  <dig> solo-ltr sequences in the vdls.
<dig> genome.
the gag proteins of these gypsy-like elements contained both gag and zinc finger conserved domains.
the pol orfs were in a − <dig> frameshift orientation relative to that of the gag orf, and comprised aspartate protease , reverse transcriptase , rnaseh, chromodomain, and integrase  domains in an organization typical of ty3/gypsy-like elements  <cit> .
the top hit from a blastp search with the  <dig> aa vdltre2/3/ <dig> pol predicted proteins was the cgret ltr element from the closely related cranberry fruit rot fungus colletotrichum gloeosporioides <cit> .
non-ltr retrotransposons of superfamily i
a line-like non-ltr retrotransposon, vdline <dig>  was identified as present in  <dig> copies.
the  <dig>  bp elements contained separate gag-like and pol orfs, and an ncbi conserved domain search identified endonuclease-exonuclease-phosphatase  and rt domains, as well as a c-terminal rnaseh domain that defines superfamily i elements  <cit> .
the top hit from a blastp search of the predicted  <dig> aa pol protein was that of a putative reverse transcriptase of p. marneffei .
“cut-and paste” transposons of superfamily tc1/mariner
among the dna transposons found in the vdls.
<dig> genome, twenty seven full-length tc1/mariner-like elements were identified by having their short, perfect inverted terminal repeats  flanked by dinucleotide ta target site duplications , and the presence between the tirs of a single intronless orf encoding a transposase characterized by the conserved endonuclease domain dde_ <dig>  <cit>  and the n-terminal dna binding domains hth_psq and hth_tnp_tc <dig> .
on the basis of tir, orf and whole te length and sequence identity, we distinguished three vdls.
<dig> tc1/mariner families referred hereafter as dahliae  <dig>   <dig> and  <dig>
among the dahliae <dig> sequences, we found  <dig> complete elements about  <dig>  kb in length.
these sequences were associated with  <dig> to 62 bp tirs that showed conservation of the first four nucleotides acgt-, and contained  <dig> to 17 bp internal direct repeats  .
we also detected about  <dig> additional defective dahliae1-like elements, five of which lack both tirs .
all dahliae <dig> predicted orfs started at te nucleotide position  <dig> and encoded  <dig> aa proteins with the dde_ <dig> endonuclease domain spanning aa positions 154– <dig> and one or both the n-terminal dna-binding domains hth_psq and hth_tnp_tc <dig> at aa positions 7– <dig> and 57– <dig>  respectively .
blastp with dahliae <dig> proteins revealed greatest sequence similarities  with sequences from ascomycete fungi, in particular, with a predicted protein from the phytopathogen f. oxysporum  .
to establish the relationships among the dahliae <dig> transposons, we performed phylogenetic analysis using the full-length nucleotide sequences, which resulted in the classification of the  <dig> subfamilies dahliae <dig> a to e .
members of the same subfamily shared the same tir length and were 95–100% identical to each other.
sequence homologies between members of the different subfamilies ranged from  <dig> to 74%.
dahliae 1a and 1d, with  <dig> and  <dig> complete copies each, respectively, were the largest subfamilies and the most abundant dna transposons in vdls.
<dig> genome.
dahliae <dig> c and e were each present in only a single complete copy, and encoded what are likely to be nonfunctional transposases due to in-frame early stop codons.
only one complete copy of dahliae <dig> was detected in vdls.
<dig>  the element was  <dig>  bp long with 62 bp tirs, and harbored an orf coding for a putative  <dig> aa polypeptide .
the tirs, like in dahliae <dig>  started with the nucleotides acgt-, comprised three mismatches, and the dr sequence tttcggacacccccccc- repeated three times at the 5’-end of the tes .
the dahliae <dig> orf started at nucleotide position  <dig>  and the encoded transposase shared the greatest overall sequence homology  with ophio <dig> from the causal agent of dutch elm disease ophiostoma ulmi <cit> .
like dahliae <dig>  only one full-length copy of dahliae <dig> was present in vdls.
<dig>  the consensus sequence was  <dig>  bp long, with 102-bp tirs.
the 5’ tir started with the sequence cccgt- and tir alignment revealed one mismatch, and lack of internal direct duplications or palindromes .
dahlaie <dig> contained a  <dig> -bp orf starting at the te nucleotide position  <dig>  and encoded a  <dig> aa transposase with similarities to a putative  <dig> aa transposase from the fungal human pathogen talaromyces stipitatus .
“cut-and paste” transposons of superfamily activator
seven vdls.
<dig> sequences, designated vdhat  <dig> to  <dig>  were found to putatively encode  <dig> to  <dig> aa hat dimerization domains, which characterize the carboxy termini of the transposases of activator-like element transposases  <cit>  .
we were able to define the consensus sequence and flanking short imperfect tirs of two single copy elements, vdhat  <dig> and  <dig>  and identify for vdhat <dig> the 8-bp tsd gttactgt containing a typical central ta-dinucleotide.
the complete vdhat  <dig> and  <dig> elements were  <dig>  and  <dig>  bp in length, with  <dig> and 20 bp tirs, respectively .
the tirs of each element comprised one mismatch and, while the vdhat <dig> tirs started with the sequence atttg-, vdhat <dig> tirs were characterized by the motif cagng- , which is also present in other fungal hat-like elements such as the cryphonectria parasitica crypt <dig>  <cit> , the f. oxysporum drifter and tfo <dig>  <cit> , and the tolypocladium inflatum restless  <cit> .
analysis of the vdhat partial and complete nucleotide sequences using blastn and clustalw revealed no significant regions of similarity to activator-like sequences from other organisms, suggesting that each vdhat was vdls.17-specific.
orf finder searches with vdhat tes and the analysis of corresponding ests supported the presence of a single transposase-coding gene.
however, the presence in most of the vdhats of in-frame early stop codons suggested that partial, likely non-functional proteins would be generated.
blastx analysis showed that while the best hits for vdhat  <dig> and  <dig> were two sequences from c. globosum, the best matches for vdhat  <dig> to  <dig> sequences were to four different predicted proteins of the recently sequenced entomopathogenic ascomycete metarhizium anisopliae <cit>  .
“cut-and paste” transposons of superfamily mutator
blastp search using the  <dig> aa mule domain from the active mutator-like element hop <dig> of f. oxysporum f. sp.
melonis <cit>  revealed the presence of eight predicted mule proteins in vdls.
<dig> .
manual inspection of the corresponding genomic sequences, which we refer to as vdmule  <dig> to  <dig>  resulted in the discovery of three potentially complete elements vdmule  <dig> ,  <dig>  and  <dig> , each encoding a single orf, and containing  <dig>   <dig>  and 53 bp tirs, respectively.
an 8 bp tsd  was associated with one element only, and defective elements ranged in size from  <dig> to  <dig>  bp .
alignment of the vdmule nucleotide sequences with hop <dig> tirs showed the presence of the terminal consensus ggnaa, and of sub-terminal drs of different lengths.
in particular, while vdmule  <dig> and  <dig> had three 15-bp and 17-bp repeats, respectively, vdmule <dig>  like hop <dig>  possessed  <dig> and 6 bp duplications .
among the vdmules, the highest nucleotide sequence homology was observed between the three full-length elements .
vdmule <dig> appeared to be the most divergent, with no significant similarity to any other vdmule element.
vdmule elements were predicted to harbor single transposase genes with different exon/intron structures.
in particular, we found a single exon in the vdmule <dig>   <dig>   <dig> and  <dig> genes, four exons in vdmule <dig>  two exons in vdmule <dig> and five exons in the vdmule <dig> genes, respectively.
complete mule domains  were present only in transposases of the full-length elements.
the remaining transposases were characterized by n- or c-terminal domain truncations of  <dig> to  <dig> aa.
notably, the predicted  <dig> aa vdmule <dig> protein  was found to be the in-frame product of the fusion between a nitrilase and a mule-transposase coding sequence.
the hypothetical protein harbors a  <dig> aa n-terminal nitrilase domain  spanning aa positions 3– <dig>  and a truncated  <dig> aa mule domain spanning aa positions 363– <dig>  vdmule <dig> transposase  architecture was also characterized by the presence of a  <dig> aa zinc-knuckle  at its carboxy terminus.
proteins with the highest sequence homology to the vdmule transposases, which ranged from 55% to 79% identity, were from c. globosum and m. anisopliae .
distribution of class i and ii tes in the vdls.
<dig> genome
an in-depth analysis of transposon distribution in vdls.
<dig> genome revealed that the most abundant elements, vdltres 1– <dig> and tc1/mariner dahliae <dig>  were dispersed throughout the genome’s eight chromosomes, and in unpositioned scaffold sequences.
however, there was notable te clustering in chromosomes  <dig> and  <dig> .
in particular, supercontings  <dig> and  <dig> of chromosome  <dig> appeared to contain “hot-spots” of te insertions, harboring 21% and 20% of the transposons, respectively.
these supercontigs also contain vdls.
<dig> ls  regions  <dig> and  <dig>  which are absent from the vams.
<dig> genome  <cit> .
the non-random te distribution of the dde_ <dig>  hat and mule motifs was further assessed, and odds ratio analyses  <cit>  did indeed show that the ls regions are significantly enriched in transposon motifs  relative to that of the total predicted gene distribution in the genome assembly of vdls.
<dig> .
one approximately  <dig> kb region from each of ls regions  <dig> and  <dig> was selected to illustrate the genetic context of some tes .
this figure highlights how the transposons are present in gene-rich locations, and shows a putative sequence duplication event involving genes encoding a chitinase and a phospholipase, in addition to those previously described  <cit> .
the verticillium albo-atrum vams.
<dig> genome contains few transposons
while tes were prevalent in the v. dahliae vdls.
<dig> genome, few repetitive sequences were identified in vams.
<dig>  blastn and blastx queries against the v. albo-atrum genome using vdls.
<dig> class i and ii transposons identified one intact, full-length element having  <dig> % sequence identity to vdltre <dig>  one full-length but degenerate copia-like element, and one partial sequence of approximately 1 kb, also of the copia-type.
only short  sequences were found that had  <dig> to  <dig> percent identity to the gypsy-like elements.
lastly, no sequences corresponding to vdltre <dig> were identified, and remarkably, neither full-length nor incomplete class ii tes were detected in the vams.
<dig> genome.
vdls.
<dig> class i and ii transposons are transcriptionally active
to assess potential transcriptional activity, we inspected the three vdls.
<dig> est libraries generated in support of the verticillium genome sequencing project  <cit>  in search of sequences corresponding to the different te types described herein.
these libraries were obtained from fungal cultures grown on control complete medium , cm supplemented with root extract from the host plant lettuce , or cm lacking nitrogen   <cit> .
we identified ests matching all of the class i and ii transposons in at least one of the growth conditions .
avdls.
<dig> expressed sequence tag  collections were generated from cultures grown in cm , cm + re , or cm-n .
for vdltre <dig>  vdltre <dig> and vdltre <dig>  the corresponding ests aligned with all three elements except that * one est aligned with only vdltre <dig>  ** four ests aligned with both vdltre  <dig> and  <dig>  and *** one est aligned with only vdltre <dig>
the est analysis showed that class i elements were transcriptionally active under all of the growth conditions.
although similar levels of expression were observed for the cm + root extract and cm -nitrogen cultures, numbers of vdltre <dig> ests were substantially higher than those of all other elements.
in contrast, within each dna te family, a differential level of expression was observed according to the fungal growth condition.
for instance, while dahliae <dig> a, d and e were transcriptionally active primarily in control and nitrogen starvation conditions, ests for dahliae <dig>  vdhat  <dig> and  <dig> and vdmule  <dig> and  <dig> were detected only in the library from cultures grown in the root extract-amended medium.
abiotic stress can stimulate expression of tes in fungi  <cit> .
we therefore used reverse transcription-pcr to make a semi-quantitative assessment of the expression of selected vdls.
<dig> dna transposases in response to a heat shock treatment .
the resulting data provided evidence that while the expression of dahliae <dig>  dahliae <dig>  vdmule <dig>  and vdmule <dig> was induced at 42°c, vdmule <dig> expression appeared to be unaffected by the treatment, and dahliae <dig> responded negatively to the treatment .
phylogenetic analyses
to gain a better understanding of the evolutionary history of the different transposable elements identified in vsls.
<dig>  we studied their phylogenetic relationships with homologous sequences from other fungal species.
analyses were done as described in the methods section and using the aa sequences of the retroelement rt domains, predicted aa sequences of the complete tc1/mariner proteins, and conserved domains of the vdhat and vdmule transposases .
consistent with the predictions based on the organization of the pol domains, vdltre <dig> and vdltre <dig> clustered with known fungal copia-like elements.
however, bootstrap support separated the two types of elements into distinct clades, with vdltre <dig> being most closely related to sequences from aspergillus capsulatus and a. terreus .
vdltres  <dig>   <dig> and  <dig> clustered together and with known gypsy elements, showing close relationships with the c. gloeosporioides element cgret  <cit>  and skippy from f. oxysporum <cit>  .
although bootstrap values supported a monophyletic origin for all the fungal dde_ <dig> transposases considered, none of the vdls.
<dig> dde_ <dig> transposases clustered with sequences from the basidiomycetes cryptococcus neoformans  and ustilago maydis , or with sequences from the four different aspergillus species included in the analysis .
while the three dahliae families, whose members share  <dig> to 34% identity, separated into  <dig> different lineages before speciation, dahliae <dig> differentiated into subfamilies, sharing  <dig> to 72% aa identity, apparently after insertion into the vdls.
<dig> genome.
both dahliae1- and dahliae2-containing clades also comprised proteins from the sordariomycetes c. globosumf.
oxysporumm.
oryzae and the leotiomycete s. sclerotiorum, which are closely related to verticillium spp.
however, dahliae  <dig> grouped with sequences from the more distantly related erotiomycete species ajellomyces capsulatusp.
marneffei, paracoccidioides brasiliensis and t. stipitatus.
among the fungal proteins clustering with dahliae <dig>  all of which possess the hth_psq/hth_tnp_tc5/dde_ <dig> domain organization, we found transposases of well characterized plant pathogen tes such as flipper of b. fuckeliana <cit> , fot <dig> of f. oxysporum <cit> , nht <dig> of nectria haematococca <cit> , and ophio  <dig> and  <dig> of o. nova-ulmi <cit> .
sequences with similarities to vdls.
<dig> hat and mule domains were identified in a much more limited number of fungi.
sequence alignments of the vdhat and vdmule domains revealed core regions of  <dig> and  <dig> aa, respectively, and led to the identification of amino acid positions that are conserved across all fungal species examined .
while the vdhats were separated into  <dig> different clades, all vdmules clustered together .
bootstrap values in the range of 50–60% or below did not support a monophyletic origin for the vdhats.
in the clade containing vdhat  <dig> and  <dig>  there were sequences from the activator-like transposons crypt <dig> of the chestnut blight fungus cryphonectria parasitica <cit>  and tfo <dig> of f. oxysporum <cit> , together with one sequence from c. globosum.
vdhat <dig> was more closely related to drifter, a hat element from f. oxysporum <cit> , and restless from the cyclosporine-producing fungus t. inflatum <cit> , as well as to sequences from c. globosumm.
anisopliae, and n. haematococca.
vdhats  <dig> to  <dig> were grouped with m. anisopliae sequences only .
the vdmule clade comprised m. anisopliae and c. globosum sequences and appeared to be unrelated to the clade with the f. oxysporum element hop and sequences from p. crysogenum, p. marneffei, and t. stipitatus .
evidence of repeat induced point  mutation in vdls.
<dig> tes
in a previous study we surveyed vdls.
<dig> retrotransponsons for evidence of rip-like mutations and found them only in gypsy-like sequences  <cit> .
here we extended the analysis to all vdls.
<dig> dna transposons.
although the sequence divergence for the activator- and mutator-like elements was significant, ripcal searches  <cit> , which included vdltre2-like sequences as positive control for riping, revealed no clear bias for rip-like mutations in any of the vdls.
<dig> “cut-and-paste” elements .
distribution of the vdls.
<dig> class i and ii tes in other v. dahliae and v. albo-atrum isolates
the distribution of the class i and ii transposons identified in vdls.
<dig> was investigated in another 21 v. dahliae and v. albo-atrum isolates from various hosts and geographic regions, including the v. dahliae vegetative compatibility group  tester strains described by joaquim and rowe  <cit>  .
the tc1/mariner dahliae <dig> showed the most widespread distribution among the two species, being present in 33% and 66% of the vd and va isolates, respectively.
the copia vdltre <dig> and the gypsy vdltr2/3/ <dig> were detected in multiple copies in almost all vd isolates, whereas the copia-like element vdltre <dig> was found only in vdls.
<dig> and four other vd isolates, and always in low copy number .
only four va isolates were conclusively positive for the presence of vdls.17-like retroelements.
the tc1/mariner dahliae <dig> and activator-like sequences were, like the retroelements, limited in their distribution, and represented primarily in v. dahliae .
lastly, tc1/mariner dahliae <dig> and vdmule elements appeared to be specific to vdls.
<dig>
the presence of the indicated class i and class ii transposable elements was determined by southern hybridization or pcr amplification using gene-specific probes  or primer pairs, respectively, as described in materials and methods and as shown in additional file 8: figure s <dig>  asequenced genomes; bvcg, vegetative compatibility groups; -, not detected; nd, no data.
discussion
we originally set out to gain a better understanding of the nature and extent of genetic diversity in phytopathogenic verticillium spp.
and, to this end identified mobile elements in the sequenced v. dahliae and v. albo-atrum genomes, and explored their distribution in other strains of different origins.
our genome-wide search yielded complete retroelements and “cut-and-paste” dna transposons, whose structure we characterized in detail.
among the vdls.
<dig> ltr sequences that we identified, the ty1/copia-like vdltre <dig> had the gag and pol orfs in an organization that is very rare  <cit> , and uses a leaky stop codon for translation of the pol orf that is downstream of the gag orf.
vdltre <dig> did not have, downstream of the gag stop codon, the conserved caryya sequence, which has been previously shown to be important for this stop codon read-through  <cit> .
however, the ty1/copia-like element tca <dig> of candida albicans also does not possess this sequence and it has been proposed that the sequences responsible for stop codon read-through of tca <dig> can be multiple, remote, and scattered throughout the element  <cit> .
the same may therefore be true for vdltre <dig>
the vdls.
<dig> retrotransposons vdltre  <dig>   <dig>  and  <dig> were identifiable as gypsy elements in their having gag and pol genes with the pol orfs in a − <dig> frameshift orientation relative to that of the gag orf  <cit> .
a notable feature differentiating these three te families, which are predicted to encode almost identical polyproteins, was the difference in their ltr lengths.
ltr elements contain critical cis-acting signals that define the element borders and act as transcriptional promoters  <cit> , and we indeed found up to 5-fold differences in the number of the ests corresponding to each type of element.
among the “cut-and-paste” tes in vdls.
<dig>  the tc1/mariner superfamily, which derives its name from the founder transposons tc <dig> of caenorhabditis elegans and mariner of drosophila mauritiana <cit> , was predominant.
all  <dig> full-length dahliae elements were approx.
<dig> kb in size and comprised single intronless dde_1-encoding transposases flanked by short  tirs.
the dde_ <dig> transposases act generally as dimers or oligomers and harbor functional domains mediating protein-protein interaction, dna-binding, -cleavage and -joining activities  <cit> .
we predicted the presence of two types of n-terminal dna-binding domains, hth_psq and hth_tnp_tc <dig>  which are involved in the recognition and interaction with the tirs  <cit> .
the dde_ <dig> domain, first identified in bacterial transposases and retroviral integrases, is characterized by conservation of three aspartate  residues or two, non-contiguous  and one glutamate  residue, a catalytic triad that forms a pocket able to bind two divalent metal ions, mostly likely mg2+, that are necessary for transposition  <cit> .
the dde signature has been detected in all eukaryotic “cut-and-paste” transposase superfamilies, indicating their common evolutionary origin  <cit> .
in the dahliae transposases, the three aspartate residues are separated by 110– <dig> and  <dig> amino acids, respectively .
in vitro and in vivo trans-kingdom assays have previously demonstrated that, while host factors are not needed for transposition, intact transposase and tirs are required for the initiation and completion of the process  <cit> .
tc1/mariner family transposon termini comprise at least three types of functional sequences involved in transposition: the 4– <dig> nucleotide te cleavage sites at the outer extremities of the tirs, the drs within the tirs, which are the transposase binding sites, and the utrs, between the tirs and orfs, which act as enhancers of transposition efficiency  <cit> .
transposons of different families generally differ in their terminus structure and length, as well as in the transposase domain architecture.
each terminus/transposase combination appears to mediate a slightly different version of the “cut-and-paste” mobilization mechanism, ensuring transposition specificity  <cit> .
in vdls.
<dig>  while dahliae <dig> starts with the unique cleavage motif cccg and does not possess recognizable repeated sequences in the tirs like those of tc1/mariner tes of other ascomycete fungi  <cit> , dahliae  <dig> and  <dig> start with the sequence acgt-, and their tirs contain two or three drs.
in particular, dahliae <dig> carries three internal repetitions of a 17 bp-sequence at the 5’ terminus, and two at the 3’ terminus.
the dahliae orfs do not overlap with the tir sequences and are flanked by asymmetric utrs that vary in length from  <dig> to 125 bp, according to the te family type.
the vdls.
<dig> activator and mutator elements are highly degenerate  or with limited sequence similarities .
most of the elements appeared to be of the non-autonomous type due to mutations that disrupted tir sequences and/or resulted in incomplete transposases.
although these elements are probably unable to transpose and are fossils, we were able to identify corresponding ests for some of them.
these sequences may therefore still play the important role of repressing transposition of the complete elements of the same family through transposase dilution or through a negative dominant repression by the truncated transposases.
domestication is the process by which te functional domains are incorporated into functional host proteins  <cit> .
in vdls.
<dig> we found an insertion of a fragment of a mutator element within a nitrilase gene.
the fused sequence is predicted to generate a protein carrying in-frame nitrilase and mule domains.
although we did not find corresponding ests, we cannot rule out the possibility that this new protein is functional.
the est data and expression analysis under heat stress further showed that several of the full length class i and ii tes, which are predicted to code for complete transposition-mediating enzymes, are still transcriptionally active and differentially responsive to different stimuli.
in general, our phylogenetic analysis of both class i and ii tes mirrored the relationships among the fungal species that were defined by the fungal genome initiative on the basis of whole genome comparative studies.
the evolutionary ancestors of the copia, dahliae, vdhat and vdmule tes apparently evolved into different groups before insertion into the vdls.
<dig> genome.
while high bootstrap values supported the monophyletic evolution of the v. dahliae tc1/mariner and mule elements, the copia vdltres  <dig> and  <dig> and the vdhats fell into distinct lineages.
the three gypsy families diversified after introgression into vdls <dig> genome.
also, the tc1/mariner family dahliae <dig> underwent a recent expansion in vdls.
<dig>  generating five vdls.17-specific subfamilies.
these subfamilies differed in sequence and length of their termini, and of their orf sequences.
dahliae <dig> a, b and d all putatively coded for intact transposases and were present in multiple, almost identical copies, comprising 74% of the total dna tes in vdls.
<dig>  in fungi, the selective amplification of transposon subfamilies, such as we have detected for the dahliae <dig> elements, has been associated to events of horizontal gene transfer  <cit> , however no definitive mechanism has yet been proven.
it has been proposed that tes may enhance recombination to cause genetic variation, giving populations the flexibility to adapt, a phenomenon which could be especially important for species that do not have a sexual phase  <cit> .
te clustering such as we observed in the vdls.
<dig> ls regions has been found in the genomes of other phytophathogenic fungi.
in m. oryzae, for example, both class i and ii transposable elements are clustered within three regions of chromosome seven, characterized by a high rate of duplication and evolution  <cit> .
similarly, tes were also found to cluster in regions undergoing rapid reorganization in the genome of the plant vascular pathogen f. oxysporum <cit> .
it has been hypothesized that these types of clusters are important for the generation and evolution of new genes  <cit> , and it has been proposed that the transposon-rich ls regions in v. dahliae may impart a degree of genetic flexibility in the species, and allow rapid adaptation to new host niches  <cit> .
the mechanism by which te clusters are generated in fungal genomes has not been elucidated yet.
while the clustering could simply be the passive result of selection against te incorporation into gene-rich zones of the genome, it alternatively could result from an active process related to te function.
many tes do selectively integrate into specific sequences  <cit> , which could lead to biased te distribution.
such selectivity has, for example, been observed for s. cerevisiae, in which ty <dig> ltr elements are most often found to be integrated into upstream regions of genes transcribed by rna polymerase iii  <cit> .
the parallel, non-random clustering of the class i and ii v. dahliae elements suggests that there may be synergistic interactions among different types of elements, and selective pressure in v. dahliae for te clustering, which in turn may be important for generating the genomic diversity necessary for niche adaptation and host range expansion.
interestingly, among the  <dig> predicted genes encoded within the ls regions, there were no “housekeeping” genes  <cit> .
rather, the predicted genes encoded proteins of potential importance in pathogenicity, including bzip transcription factors, ferric reductases, phospholipases, and other genes predicted to play a role in response to stress  <cit> .
moreover, clusters or pairs of genes were clearly duplicated in these ls regions .
it is unclear if such duplications are the direct result of te activity, and more studies are required to investigate the role of these putative pathogenicity factors.
however, the presence of solo-ltr sequences does suggest that recombination between the repeated sequences of the ltr elements could be a contributing factor in the reorganization of verticillium dahliae genome.
since tes can have a large effect on the genomes of their hosts, causing gene deletion and duplication as well as chromosomal rearrangements, host fitness could be adversely affected if te-induced transposition and recombination events disrupted or altered function of essential genes.
however, some filamentous fungi have a unique tool, known as repeat-induced point mutation , to deal with repetitive sequences such as tes.
rip, a process first described in neurospora crassa <cit> , was found to occur during the sexual cycle, and to subject duplicated sequences of > <dig> base pairs to irreversible cg to ta transition point mutations.
although rip is known to only occur during the fungal sexual cycle, there are several examples in asexual fungi where tes with rip-like mutations have been identified  <cit> .
the identification of rip-like mutations in some vdltre2/3/ <dig> sequences  <cit>  indicated that, at some point in verticillium spp.
evolution, a rip-like process operated to protect the genome from infiltration by tes.
the presence in v. dahliae and v. albo-atrum of identifiable rid-like protein orthologs, which are known to be a part of the rip machinery in n. crassa <cit> , suggests that these fungi may still possess the capacity for rip.
interestingly, in vdls.
<dig>  rip mutations affected members of the gypsy but not copia superfamilies of retrotransposons, indicating either a differential susceptibility to rip by different types of tes or introgression by horizontal transfer of already riped sequences  <cit> .
the te content of different organisms is variable, sometimes accounting for as much as 60–80% of the genome, as in the case of cereals.
more recently, mobile elements have been found to account for about half of the genome size also in the phytopathogenic oomycete phytophthora infestans and the truffle fungus tuber melanosporum <cit> .
noticeably, a high degree of variability in the number and types of transposons has been documented even in closely related species.
for instance, the te content of the fungal plant pathogens f. oxysporum and f. graminearum differs by a two-fold order of magnitude, 4% and  <dig> %, respectively  <cit> .
the observed te spatial-temporal fluctuations appear to be te-family dependent and governed by multiple mechanisms including elimination by ectopic recombination, extinction by genetic drift, reintroduction by horizontal transfer, and environmental stress-driven expansion .
despite the high level of identity  and synteny identified between the genomes of the recently sequenced vd and va isolates, the v. albo-atrum genome assembly was distinct in containing far less repetitive dna than did that of v. dahliae <cit> .
in particular, vams.
<dig> lacked the highly repetitive ls regions present in vdls.
<dig>  and contained neither full-length nor defective vdltre <dig> copia or “cut-and-paste” dna elements.
this absence seems simply to reflect the isolate of v. albo-atrum sequenced rather than a general feature of the species.
in fact, 66% of the other va isolates we surveyed for the presence of vd.ls <dig> te-like sequences were positive for the tc1/mariner dahliae <dig>
extensive studies conducted on natural populations of other organisms such as drosophila and different plant species including arabidopsis, barley, maize, rice and wheat have clearly demonstrated that transposon dynamics plays a central role also in generating intraspecific variability  <cit> .
our findings have shown that the copia-like vdltre <dig> and the gypsy-like retrotransposons are almost ubiquitous in v. dahliae, whereas the copia-like vdltre <dig> and most of the “cut-and-paste” dna tes appear to have a much more limited distribution.
although lack of detection of vdls.17-like tes could indeed reflect total absence of mobile elements, those verticillium genomes without such elements may either contain related sequences that have diverged to a degree that prevented detection under the conditions used in this study , or may simply harbor their own distinct arrays of transposable elements.
CONCLUSIONS
the identification and characterization of class i and ii tes of verticillium dahliae  has allowed further exploration of the genetic diversity existing among the phytopathogenic verticillium spp., and raised intriguing questions about the role that tes may have in shaping intra- and inter-specific evolution in this fungal genus.
the discovery of chromosome location “hot spots” for te insertion, as well as a “patchy” distribution among isolates of some of the tes offers tantalizing evidence that tes may play an important role in modulating genome architecture to allow verticillium fungi to evolve quickly and adapt to their hosts, as has been suggested for other plant pathogens  <cit> .
v. dahliae and v. albo-atrum were previously perceived, on the basis of vegetative compatibility and pathogenicity analyses, to have rather low genetic diversity.
however, the results of molecular analyses carried out over the past two decades suggest that the species do in fact exhibit a high level of genetic variation and genome plasticity  <cit> .
the analyses presented herein showed a broad range of different combinations of te types in the different fungal strains, further supporting a major contribution of the transposable elements to the diversification of verticillium spp.
genomes.

 present in the environment as free-living organisms or in symbiosis with other organisms, fungi have a major impact on human society. besides being major contributors to the decomposition and recycling of nutrients, they are largely employed in food production, biotechnologies and agricultural systems. however, these microorganisms also cause devastating diseases, particularly of plants, representing a major threat to global food security. it is noteworthy that a significant number of plant pathogenic fungi are able to cause serious human and animal diseases, for which there are very few effective therapeutic agents. verticillium dahliae and v. albo-atrum cause very disruptive vascular diseases in over plant species, including vegetable, ornamental and tree crops. these microorganisms penetrate the root system of their hosts and induce typical leaf wilt symptoms by spreading throughout the xylem vessels and disrupting water transport. compared to va, vd is generally characterized by a broader host range and geographical distribution, and the capability to produce longer lasting soil resting structures called microsclerotia. no sexual reproduction has been reported in either vd or va, thus implicating somatic mutation as a driving force in their evolution. comparative analysis of the recently sequenced genomes of the isolates vdls. and vams., showed that they are 97% identical and that vdls. contains regions in chromosomes three and four that are not present in the va isolate. these lineage-specific regions, which encompass about % of the fungal genome, comprise about protein-coding genes and are also enriched in repetitive sequences corresponding to both previously known and as-yet uncharacterized transposon-like sequences. transposable elements are found in virtually every prokaryotic and eukaryotic genome where they can exert a multifaceted mutagenic activity that leads to changes in chromosome architecture, generation of new regulatory networks and increases in the protein repertoire. on the basis of structural-functional characteristics, tes have been separated into two major classes each comprising subclasses or orders, superfamilies, families and subfamilies. the elements of class i, retrotransposons, replicate by a “copy-and-paste” mechanism that generates rna intermediates, which are subsequently reverse transcribed into double-stranded dna by te-encoded enzymes. these retroelements have been recently classified into five orders comprised of long terminal repeat elements, dictyostelium intermediate repeat sequence, penelope-like elements, and long and short interspersed nuclear elements. among the most widespread retrotransposons are the ltr superfamilies gypsy and copia, which include two orfs coding for the structural virus-like protein gag and the reverse transcriptase /integrase enzyme pol. class ii elements, dna transposons, are mobilized via the formation of dna intermediates. if the intermediate is dsdna, the transposition is called “conservative” and the elements are of the “cut-and-paste” type. complete, alias autonomous, tes of this type are generally delimited by terminal inverted repeats and encode a single transposase. the transposase binds conserved regions within the tirs, mostly direct short tandem duplications, and catalyzes the excision of the complete element, which then reintegrates in a new genomic location. when te mobilization proceeds via single-stranded dna and does not involve the element excision it is defined as “replicative”, a mode of transposition that characterizes the more complex helitron- and maverick-like transposons. although fungal genomes generally contain fewer repetitive sequences than do higher eukaryotes, tes have played critical roles in the evolution of their fungal hosts and, in particular, of the phytopathogens. in the rice blast fungus, magnaporthe oryzae, for example, tes were found to cluster in regions having high recombination rates and gene duplication events. similarly, in the black mold aspergillus niger, specific retrotransposon-mediated recombinations have led to inversions of genomic regions. in addition, host-specific toxin genes of cochliobolus carbonum and alternaria alternata, as well as host-specificity genes of m. oryzae are situated in transposon-rich regions of the genome. because gene products conferring host-specificity can be recognized by plant host defense receptors, it has been hypothesized that this proximity could allow expansion of host range through te-mediated gene loss. in addition, te distribution can be extremely variable between isolates of a single fungal species, and tes have been used as markers to distinguish genetically divergent populations, and for the identification of subpopulations. introgression of mobile elements into the genome is potentially deleterious, and cells have evolved different processes for their elimination, including silencing by rna interference and repeat-induced point mutation. the introduction of rip mutations in the dna of fungi is linked to sexual reproduction, occurring at the time of meiosis, and the process likely arose as a genome defense mechanism against the intrusion of repetitive dna sequences. the rip machinery recognizes duplicated sequences longer than 400 bp and sharing more than 80% identity, and introduces in both copies c:g to t:a transitions. noticeably, rip has been detected in the asexual fungi a. nigerpenicillium crysogenum and v. dahliae, suggesting the existence of an ancestral or cryptic sexual state in these species. interestingly, rip mutations in vdls. affected members of the gypsy but not of the copia superfamilies of retrotransposons, indicating either a differential susceptibility to rip by different types of tes, or introgression of already riped sequences from a sexual organism. to further investigate the nature and extent of genetic diversity in v. dahliae, we have undertaken an analysis of tes in this species. we report here the identification and characterization of class i and ii transposons in v. dahliae, an analysis of their distribution in v. dahliae and v. albo-atrum, and discuss their potential role as generators of diversity in the verticillium genomes.  transposable elements in verticillium dahliae genome using a combination of bioinformatics predictions and manual inspections, we identified complete class i retrotransposons and class ii “cut-and-paste” dna transposons in the vdls. genome. based on comparison to consensus sequence structures, the retrotransposons were identified as members of the ltr class copia and gypsy superfamilies, and line superfamily i, and the dna tes as subclass i members of the tir superfamilies tc1/mariner, activator and mutator. ltr retrotransposons of superfamily copia we identified full-length copia-like tes, referred to as vdltre and respectively. the copies of vdltre were  bp in length, flanked by 213 bp ltrs, and contained a single orf with fused gag and pol sequences predicted to encode a amino acid polyprotein. in contrast, the two copies of vdltre had shorter ltrs, and gag and pol orfs separated by a uga stop codon. this rarely observed orf organization requires a leaky stop codon for translation read-through of the pol orf 3’ into the gag orf. a conserved caryya sequence has been shown to be important for the stop codon read-through, however a search of vdltre showed that it lacked this canonical sequence motif. a motif search identified in both vdltre and vdltre an integrase core domain located n-terminally to a reverse transcriptase domain, placing both elements as members of the ty1/copia family of retroelements. these retrotransposases shared the highest sequences similarities with hypothetical proteins of the fungal animal pathogen penicillium marneffei. finally, a genome-wide search was conducted that identified two vdltre solo-ltrs. such sequences are commonly observed remnants of ltr retrotransposons, and likely arise from recombination between the direct repeat sequences that flank retroelements. ltr retrotransposons of superfamily gypsy three families of gypsy-like elements were discovered and referred to as vdltre vdltre and vdltre, and corresponded to vdf and vdf previously identified as v. dahliae retrotransposon-like sequences. these elements were ~  to kb in size, % identical at the nucleotide level, encoded nearly identical gag and pol proteins, and possessed ltrs of and 455 bp, respectively. there were full-length elements and solo-ltr sequences in the vdls. genome. the gag proteins of these gypsy-like elements contained both gag and zinc finger conserved domains. the pol orfs were in a − frameshift orientation relative to that of the gag orf, and comprised aspartate protease, reverse transcriptase, rnaseh, chromodomain, and integrase domains in an organization typical of ty3/gypsy-like elements. the top hit from a blastp search with the aa vdltre2/3/ pol predicted proteins was the cgret ltr element from the closely related cranberry fruit rot fungus colletotrichum gloeosporioides. non-ltr retrotransposons of superfamily i a line-like non-ltr retrotransposon, vdline was identified as present in copies. the  bp elements contained separate gag-like and pol orfs, and an ncbi conserved domain search identified endonuclease-exonuclease-phosphatase and rt domains, as well as a c-terminal rnaseh domain that defines superfamily i elements. the top hit from a blastp search of the predicted aa pol protein was that of a putative reverse transcriptase of p. marneffei. “cut-and paste” transposons of superfamily tc1/mariner among the dna transposons found in the vdls. genome, twenty seven full-length tc1/mariner-like elements were identified by having their short, perfect inverted terminal repeats flanked by dinucleotide ta target site duplications, and the presence between the tirs of a single intronless orf encoding a transposase characterized by the conserved endonuclease domain dde_ and the n-terminal dna binding domains hth_psq and hth_tnp_tc. on the basis of tir, orf and whole te length and sequence identity, we distinguished three vdls. tc1/mariner families referred hereafter as dahliae and among the dahliae sequences, we found complete elements about  kb in length. these sequences were associated with to 62 bp tirs that showed conservation of the first four nucleotides acgt-, and contained to 17 bp internal direct repeats. we also detected about additional defective dahliae1-like elements, five of which lack both tirs. all dahliae predicted orfs started at te nucleotide position and encoded aa proteins with the dde_ endonuclease domain spanning aa positions 154– and one or both the n-terminal dna-binding domains hth_psq and hth_tnp_tc at aa positions 7– and 57– respectively. blastp with dahliae proteins revealed greatest sequence similarities with sequences from ascomycete fungi, in particular, with a predicted protein from the phytopathogen f. oxysporum. to establish the relationships among the dahliae transposons, we performed phylogenetic analysis using the full-length nucleotide sequences, which resulted in the classification of the subfamilies dahliae a to e. members of the same subfamily shared the same tir length and were 95–100% identical to each other. sequence homologies between members of the different subfamilies ranged from to 74%. dahliae 1a and 1d, with and complete copies each, respectively, were the largest subfamilies and the most abundant dna transposons in vdls. genome. dahliae c and e were each present in only a single complete copy, and encoded what are likely to be nonfunctional transposases due to in-frame early stop codons. only one complete copy of dahliae was detected in vdls. the element was  bp long with 62 bp tirs, and harbored an orf coding for a putative aa polypeptide. the tirs, like in dahliae started with the nucleotides acgt-, comprised three mismatches, and the dr sequence tttcggacacccccccc- repeated three times at the 5’-end of the tes. the dahliae orf started at nucleotide position and the encoded transposase shared the greatest overall sequence homology with ophio from the causal agent of dutch elm disease ophiostoma ulmi. like dahliae only one full-length copy of dahliae was present in vdls. the consensus sequence was  bp long, with 102-bp tirs. the 5’ tir started with the sequence cccgt- and tir alignment revealed one mismatch, and lack of internal direct duplications or palindromes. dahlaie contained a -bp orf starting at the te nucleotide position and encoded a aa transposase with similarities to a putative aa transposase from the fungal human pathogen talaromyces stipitatus. “cut-and paste” transposons of superfamily activator seven vdls. sequences, designated vdhat to were found to putatively encode to aa hat dimerization domains, which characterize the carboxy termini of the transposases of activator-like element transposases. we were able to define the consensus sequence and flanking short imperfect tirs of two single copy elements, vdhat and and identify for vdhat the 8-bp tsd gttactgt containing a typical central ta-dinucleotide. the complete vdhat and elements were and  bp in length, with and 20 bp tirs, respectively. the tirs of each element comprised one mismatch and, while the vdhat tirs started with the sequence atttg-, vdhat tirs were characterized by the motif cagng-, which is also present in other fungal hat-like elements such as the cryphonectria parasitica crypt, the f. oxysporum drifter and tfo, and the tolypocladium inflatum restless. analysis of the vdhat partial and complete nucleotide sequences using blastn and clustalw revealed no significant regions of similarity to activator-like sequences from other organisms, suggesting that each vdhat was vdls.17-specific. orf finder searches with vdhat tes and the analysis of corresponding ests supported the presence of a single transposase-coding gene. however, the presence in most of the vdhats of in-frame early stop codons suggested that partial, likely non-functional proteins would be generated. blastx analysis showed that while the best hits for vdhat and were two sequences from c. globosum, the best matches for vdhat to sequences were to four different predicted proteins of the recently sequenced entomopathogenic ascomycete metarhizium anisopliae. “cut-and paste” transposons of superfamily mutator blastp search using the aa mule domain from the active mutator-like element hop of f. oxysporum f. sp. melonis revealed the presence of eight predicted mule proteins in vdls.. manual inspection of the corresponding genomic sequences, which we refer to as vdmule to resulted in the discovery of three potentially complete elements vdmule, and, each encoding a single orf, and containing and 53 bp tirs, respectively. an 8 bp tsd was associated with one element only, and defective elements ranged in size from to  bp. alignment of the vdmule nucleotide sequences with hop tirs showed the presence of the terminal consensus ggnaa, and of sub-terminal drs of different lengths. in particular, while vdmule and had three 15-bp and 17-bp repeats, respectively, vdmule like hop possessed and 6 bp duplications. among the vdmules, the highest nucleotide sequence homology was observed between the three full-length elements. vdmule appeared to be the most divergent, with no significant similarity to any other vdmule element. vdmule elements were predicted to harbor single transposase genes with different exon/intron structures. in particular, we found a single exon in the vdmule and genes, four exons in vdmule two exons in vdmule and five exons in the vdmule genes, respectively. complete mule domains were present only in transposases of the full-length elements. the remaining transposases were characterized by n- or c-terminal domain truncations of to aa. notably, the predicted aa vdmule protein was found to be the in-frame product of the fusion between a nitrilase and a mule-transposase coding sequence. the hypothetical protein harbors a aa n-terminal nitrilase domain spanning aa positions 3– and a truncated aa mule domain spanning aa positions 363– vdmule transposase architecture was also characterized by the presence of a aa zinc-knuckle at its carboxy terminus. proteins with the highest sequence homology to the vdmule transposases, which ranged from 55% to 79% identity, were from c. globosum and m. anisopliae. distribution of class i and ii tes in the vdls. genome an in-depth analysis of transposon distribution in vdls. genome revealed that the most abundant elements, vdltres 1– and tc1/mariner dahliae were dispersed throughout the genome’s eight chromosomes, and in unpositioned scaffold sequences. however, there was notable te clustering in chromosomes and. in particular, supercontings and of chromosome appeared to contain “hot-spots” of te insertions, harboring 21% and 20% of the transposons, respectively. these supercontigs also contain vdls. ls regions and which are absent from the vams. genome. the non-random te distribution of the dde_ hat and mule motifs was further assessed, and odds ratio analyses did indeed show that the ls regions are significantly enriched in transposon motifs relative to that of the total predicted gene distribution in the genome assembly of vdls.. one approximately kb region from each of ls regions and was selected to illustrate the genetic context of some tes. this figure highlights how the transposons are present in gene-rich locations, and shows a putative sequence duplication event involving genes encoding a chitinase and a phospholipase, in addition to those previously described. the verticillium albo-atrum vams. genome contains few transposons while tes were prevalent in the v. dahliae vdls. genome, few repetitive sequences were identified in vams. blastn and blastx queries against the v. albo-atrum genome using vdls. class i and ii transposons identified one intact, full-length element having % sequence identity to vdltre one full-length but degenerate copia-like element, and one partial sequence of approximately 1 kb, also of the copia-type. only short sequences were found that had to percent identity to the gypsy-like elements. lastly, no sequences corresponding to vdltre were identified, and remarkably, neither full-length nor incomplete class ii tes were detected in the vams. genome. vdls. class i and ii transposons are transcriptionally active to assess potential transcriptional activity, we inspected the three vdls. est libraries generated in support of the verticillium genome sequencing project in search of sequences corresponding to the different te types described herein. these libraries were obtained from fungal cultures grown on control complete medium, cm supplemented with root extract from the host plant lettuce, or cm lacking nitrogen. we identified ests matching all of the class i and ii transposons in at least one of the growth conditions. avdls. expressed sequence tag collections were generated from cultures grown in cm, cm + re, or cm-n. for vdltre vdltre and vdltre the corresponding ests aligned with all three elements except that * one est aligned with only vdltre ** four ests aligned with both vdltre and and *** one est aligned with only vdltre the est analysis showed that class i elements were transcriptionally active under all of the growth conditions. although similar levels of expression were observed for the cm + root extract and cm -nitrogen cultures, numbers of vdltre ests were substantially higher than those of all other elements. in contrast, within each dna te family, a differential level of expression was observed according to the fungal growth condition. for instance, while dahliae a, d and e were transcriptionally active primarily in control and nitrogen starvation conditions, ests for dahliae vdhat and and vdmule and were detected only in the library from cultures grown in the root extract-amended medium. abiotic stress can stimulate expression of tes in fungi. we therefore used reverse transcription-pcr to make a semi-quantitative assessment of the expression of selected vdls. dna transposases in response to a heat shock treatment. the resulting data provided evidence that while the expression of dahliae dahliae vdmule and vdmule was induced at 42°c, vdmule expression appeared to be unaffected by the treatment, and dahliae responded negatively to the treatment. phylogenetic analyses to gain a better understanding of the evolutionary history of the different transposable elements identified in vsls. we studied their phylogenetic relationships with homologous sequences from other fungal species. analyses were done as described in the methods section and using the aa sequences of the retroelement rt domains, predicted aa sequences of the complete tc1/mariner proteins, and conserved domains of the vdhat and vdmule transposases. consistent with the predictions based on the organization of the pol domains, vdltre and vdltre clustered with known fungal copia-like elements. however, bootstrap support separated the two types of elements into distinct clades, with vdltre being most closely related to sequences from aspergillus capsulatus and a. terreus. vdltres and clustered together and with known gypsy elements, showing close relationships with the c. gloeosporioides element cgret and skippy from f. oxysporum. although bootstrap values supported a monophyletic origin for all the fungal dde_ transposases considered, none of the vdls. dde_ transposases clustered with sequences from the basidiomycetes cryptococcus neoformans and ustilago maydis, or with sequences from the four different aspergillus species included in the analysis. while the three dahliae families, whose members share to 34% identity, separated into different lineages before speciation, dahliae differentiated into subfamilies, sharing to 72% aa identity, apparently after insertion into the vdls. genome. both dahliae1- and dahliae2-containing clades also comprised proteins from the sordariomycetes c. globosumf. oxysporumm. oryzae and the leotiomycete s. sclerotiorum, which are closely related to verticillium spp. however, dahliae grouped with sequences from the more distantly related erotiomycete species ajellomyces capsulatusp. marneffei, paracoccidioides brasiliensis and t. stipitatus. among the fungal proteins clustering with dahliae all of which possess the hth_psq/hth_tnp_tc5/dde_ domain organization, we found transposases of well characterized plant pathogen tes such as flipper of b. fuckeliana, fot of f. oxysporum, nht of nectria haematococca, and ophio and of o. nova-ulmi. sequences with similarities to vdls. hat and mule domains were identified in a much more limited number of fungi. sequence alignments of the vdhat and vdmule domains revealed core regions of and aa, respectively, and led to the identification of amino acid positions that are conserved across all fungal species examined. while the vdhats were separated into different clades, all vdmules clustered together. bootstrap values in the range of 50–60% or below did not support a monophyletic origin for the vdhats. in the clade containing vdhat and there were sequences from the activator-like transposons crypt of the chestnut blight fungus cryphonectria parasitica and tfo of f. oxysporum, together with one sequence from c. globosum. vdhat was more closely related to drifter, a hat element from f. oxysporum, and restless from the cyclosporine-producing fungus t. inflatum, as well as to sequences from c. globosumm. anisopliae, and n. haematococca. vdhats to were grouped with m. anisopliae sequences only. the vdmule clade comprised m. anisopliae and c. globosum sequences and appeared to be unrelated to the clade with the f. oxysporum element hop and sequences from p. crysogenum, p. marneffei, and t. stipitatus. evidence of repeat induced point mutation in vdls. tes in a previous study we surveyed vdls. retrotransponsons for evidence of rip-like mutations and found them only in gypsy-like sequences. here we extended the analysis to all vdls. dna transposons. although the sequence divergence for the activator- and mutator-like elements was significant, ripcal searches, which included vdltre2-like sequences as positive control for riping, revealed no clear bias for rip-like mutations in any of the vdls. “cut-and-paste” elements. distribution of the vdls. class i and ii tes in other v. dahliae and v. albo-atrum isolates the distribution of the class i and ii transposons identified in vdls. was investigated in another 21 v. dahliae and v. albo-atrum isolates from various hosts and geographic regions, including the v. dahliae vegetative compatibility group tester strains described by joaquim and rowe. the tc1/mariner dahliae showed the most widespread distribution among the two species, being present in 33% and 66% of the vd and va isolates, respectively. the copia vdltre and the gypsy vdltr2/3/ were detected in multiple copies in almost all vd isolates, whereas the copia-like element vdltre was found only in vdls. and four other vd isolates, and always in low copy number. only four va isolates were conclusively positive for the presence of vdls.17-like retroelements. the tc1/mariner dahliae and activator-like sequences were, like the retroelements, limited in their distribution, and represented primarily in v. dahliae. lastly, tc1/mariner dahliae and vdmule elements appeared to be specific to vdls. the presence of the indicated class i and class ii transposable elements was determined by southern hybridization or pcr amplification using gene-specific probes or primer pairs, respectively, as described in materials and methods and as shown in additional file 8: figure s asequenced genomes; bvcg, vegetative compatibility groups; -, not detected; nd, no data. discussion we originally set out to gain a better understanding of the nature and extent of genetic diversity in phytopathogenic verticillium spp. and, to this end identified mobile elements in the sequenced v. dahliae and v. albo-atrum genomes, and explored their distribution in other strains of different origins. our genome-wide search yielded complete retroelements and “cut-and-paste” dna transposons, whose structure we characterized in detail. among the vdls. ltr sequences that we identified, the ty1/copia-like vdltre had the gag and pol orfs in an organization that is very rare, and uses a leaky stop codon for translation of the pol orf that is downstream of the gag orf. vdltre did not have, downstream of the gag stop codon, the conserved caryya sequence, which has been previously shown to be important for this stop codon read-through. however, the ty1/copia-like element tca of candida albicans also does not possess this sequence and it has been proposed that the sequences responsible for stop codon read-through of tca can be multiple, remote, and scattered throughout the element. the same may therefore be true for vdltre the vdls. retrotransposons vdltre and were identifiable as gypsy elements in their having gag and pol genes with the pol orfs in a − frameshift orientation relative to that of the gag orf. a notable feature differentiating these three te families, which are predicted to encode almost identical polyproteins, was the difference in their ltr lengths. ltr elements contain critical cis-acting signals that define the element borders and act as transcriptional promoters, and we indeed found up to 5-fold differences in the number of the ests corresponding to each type of element. among the “cut-and-paste” tes in vdls. the tc1/mariner superfamily, which derives its name from the founder transposons tc of caenorhabditis elegans and mariner of drosophila mauritiana, was predominant. all full-length dahliae elements were approx. kb in size and comprised single intronless dde_1-encoding transposases flanked by short tirs. the dde_ transposases act generally as dimers or oligomers and harbor functional domains mediating protein-protein interaction, dna-binding, -cleavage and -joining activities. we predicted the presence of two types of n-terminal dna-binding domains, hth_psq and hth_tnp_tc which are involved in the recognition and interaction with the tirs. the dde_ domain, first identified in bacterial transposases and retroviral integrases, is characterized by conservation of three aspartate residues or two, non-contiguous and one glutamate residue, a catalytic triad that forms a pocket able to bind two divalent metal ions, mostly likely mg2+, that are necessary for transposition. the dde signature has been detected in all eukaryotic “cut-and-paste” transposase superfamilies, indicating their common evolutionary origin. in the dahliae transposases, the three aspartate residues are separated by 110– and amino acids, respectively. in vitro and in vivo trans-kingdom assays have previously demonstrated that, while host factors are not needed for transposition, intact transposase and tirs are required for the initiation and completion of the process. tc1/mariner family transposon termini comprise at least three types of functional sequences involved in transposition: the 4– nucleotide te cleavage sites at the outer extremities of the tirs, the drs within the tirs, which are the transposase binding sites, and the utrs, between the tirs and orfs, which act as enhancers of transposition efficiency. transposons of different families generally differ in their terminus structure and length, as well as in the transposase domain architecture. each terminus/transposase combination appears to mediate a slightly different version of the “cut-and-paste” mobilization mechanism, ensuring transposition specificity. in vdls. while dahliae starts with the unique cleavage motif cccg and does not possess recognizable repeated sequences in the tirs like those of tc1/mariner tes of other ascomycete fungi, dahliae and start with the sequence acgt-, and their tirs contain two or three drs. in particular, dahliae carries three internal repetitions of a 17 bp-sequence at the 5’ terminus, and two at the 3’ terminus. the dahliae orfs do not overlap with the tir sequences and are flanked by asymmetric utrs that vary in length from to 125 bp, according to the te family type. the vdls. activator and mutator elements are highly degenerate or with limited sequence similarities. most of the elements appeared to be of the non-autonomous type due to mutations that disrupted tir sequences and/or resulted in incomplete transposases. although these elements are probably unable to transpose and are fossils, we were able to identify corresponding ests for some of them. these sequences may therefore still play the important role of repressing transposition of the complete elements of the same family through transposase dilution or through a negative dominant repression by the truncated transposases. domestication is the process by which te functional domains are incorporated into functional host proteins. in vdls. we found an insertion of a fragment of a mutator element within a nitrilase gene. the fused sequence is predicted to generate a protein carrying in-frame nitrilase and mule domains. although we did not find corresponding ests, we cannot rule out the possibility that this new protein is functional. the est data and expression analysis under heat stress further showed that several of the full length class i and ii tes, which are predicted to code for complete transposition-mediating enzymes, are still transcriptionally active and differentially responsive to different stimuli. in general, our phylogenetic analysis of both class i and ii tes mirrored the relationships among the fungal species that were defined by the fungal genome initiative on the basis of whole genome comparative studies. the evolutionary ancestors of the copia, dahliae, vdhat and vdmule tes apparently evolved into different groups before insertion into the vdls. genome. while high bootstrap values supported the monophyletic evolution of the v. dahliae tc1/mariner and mule elements, the copia vdltres and and the vdhats fell into distinct lineages. the three gypsy families diversified after introgression into vdls genome. also, the tc1/mariner family dahliae underwent a recent expansion in vdls. generating five vdls.17-specific subfamilies. these subfamilies differed in sequence and length of their termini, and of their orf sequences. dahliae a, b and d all putatively coded for intact transposases and were present in multiple, almost identical copies, comprising 74% of the total dna tes in vdls. in fungi, the selective amplification of transposon subfamilies, such as we have detected for the dahliae elements, has been associated to events of horizontal gene transfer, however no definitive mechanism has yet been proven. it has been proposed that tes may enhance recombination to cause genetic variation, giving populations the flexibility to adapt, a phenomenon which could be especially important for species that do not have a sexual phase. te clustering such as we observed in the vdls. ls regions has been found in the genomes of other phytophathogenic fungi. in m. oryzae, for example, both class i and ii transposable elements are clustered within three regions of chromosome seven, characterized by a high rate of duplication and evolution. similarly, tes were also found to cluster in regions undergoing rapid reorganization in the genome of the plant vascular pathogen f. oxysporum. it has been hypothesized that these types of clusters are important for the generation and evolution of new genes, and it has been proposed that the transposon-rich ls regions in v. dahliae may impart a degree of genetic flexibility in the species, and allow rapid adaptation to new host niches. the mechanism by which te clusters are generated in fungal genomes has not been elucidated yet. while the clustering could simply be the passive result of selection against te incorporation into gene-rich zones of the genome, it alternatively could result from an active process related to te function. many tes do selectively integrate into specific sequences, which could lead to biased te distribution. such selectivity has, for example, been observed for s. cerevisiae, in which ty ltr elements are most often found to be integrated into upstream regions of genes transcribed by rna polymerase iii. the parallel, non-random clustering of the class i and ii v. dahliae elements suggests that there may be synergistic interactions among different types of elements, and selective pressure in v. dahliae for te clustering, which in turn may be important for generating the genomic diversity necessary for niche adaptation and host range expansion. interestingly, among the predicted genes encoded within the ls regions, there were no “housekeeping” genes. rather, the predicted genes encoded proteins of potential importance in pathogenicity, including bzip transcription factors, ferric reductases, phospholipases, and other genes predicted to play a role in response to stress. moreover, clusters or pairs of genes were clearly duplicated in these ls regions. it is unclear if such duplications are the direct result of te activity, and more studies are required to investigate the role of these putative pathogenicity factors. however, the presence of solo-ltr sequences does suggest that recombination between the repeated sequences of the ltr elements could be a contributing factor in the reorganization of verticillium dahliae genome. since tes can have a large effect on the genomes of their hosts, causing gene deletion and duplication as well as chromosomal rearrangements, host fitness could be adversely affected if te-induced transposition and recombination events disrupted or altered function of essential genes. however, some filamentous fungi have a unique tool, known as repeat-induced point mutation, to deal with repetitive sequences such as tes. rip, a process first described in neurospora crassa, was found to occur during the sexual cycle, and to subject duplicated sequences of > base pairs to irreversible cg to ta transition point mutations. although rip is known to only occur during the fungal sexual cycle, there are several examples in asexual fungi where tes with rip-like mutations have been identified. the identification of rip-like mutations in some vdltre2/3/ sequences indicated that, at some point in verticillium spp. evolution, a rip-like process operated to protect the genome from infiltration by tes. the presence in v. dahliae and v. albo-atrum of identifiable rid-like protein orthologs, which are known to be a part of the rip machinery in n. crassa, suggests that these fungi may still possess the capacity for rip. interestingly, in vdls. rip mutations affected members of the gypsy but not copia superfamilies of retrotransposons, indicating either a differential susceptibility to rip by different types of tes or introgression by horizontal transfer of already riped sequences. the te content of different organisms is variable, sometimes accounting for as much as 60–80% of the genome, as in the case of cereals. more recently, mobile elements have been found to account for about half of the genome size also in the phytopathogenic oomycete phytophthora infestans and the truffle fungus tuber melanosporum. noticeably, a high degree of variability in the number and types of transposons has been documented even in closely related species. for instance, the te content of the fungal plant pathogens f. oxysporum and f. graminearum differs by a two-fold order of magnitude, 4% and %, respectively. the observed te spatial-temporal fluctuations appear to be te-family dependent and governed by multiple mechanisms including elimination by ectopic recombination, extinction by genetic drift, reintroduction by horizontal transfer, and environmental stress-driven expansion. despite the high level of identity and synteny identified between the genomes of the recently sequenced vd and va isolates, the v. albo-atrum genome assembly was distinct in containing far less repetitive dna than did that of v. dahliae. in particular, vams. lacked the highly repetitive ls regions present in vdls. and contained neither full-length nor defective vdltre copia or “cut-and-paste” dna elements. this absence seems simply to reflect the isolate of v. albo-atrum sequenced rather than a general feature of the species. in fact, 66% of the other va isolates we surveyed for the presence of vd.ls te-like sequences were positive for the tc1/mariner dahliae extensive studies conducted on natural populations of other organisms such as drosophila and different plant species including arabidopsis, barley, maize, rice and wheat have clearly demonstrated that transposon dynamics plays a central role also in generating intraspecific variability. our findings have shown that the copia-like vdltre and the gypsy-like retrotransposons are almost ubiquitous in v. dahliae, whereas the copia-like vdltre and most of the “cut-and-paste” dna tes appear to have a much more limited distribution. although lack of detection of vdls.17-like tes could indeed reflect total absence of mobile elements, those verticillium genomes without such elements may either contain related sequences that have diverged to a degree that prevented detection under the conditions used in this study, or may simply harbor their own distinct arrays of transposable elements.  the identification and characterization of class i and ii tes of verticillium dahliae has allowed further exploration of the genetic diversity existing among the phytopathogenic verticillium spp., and raised intriguing questions about the role that tes may have in shaping intra- and inter-specific evolution in this fungal genus. the discovery of chromosome location “hot spots” for te insertion, as well as a “patchy” distribution among isolates of some of the tes offers tantalizing evidence that tes may play an important role in modulating genome architecture to allow verticillium fungi to evolve quickly and adapt to their hosts, as has been suggested for other plant pathogens. v. dahliae and v. albo-atrum were previously perceived, on the basis of vegetative compatibility and pathogenicity analyses, to have rather low genetic diversity. however, the results of molecular analyses carried out over the past two decades suggest that the species do in fact exhibit a high level of genetic variation and genome plasticity. the analyses presented herein showed a broad range of different combinations of te types in the different fungal strains, further supporting a major contribution of the transposable elements to the diversification of verticillium spp. genomes. 
the observed biased te insertion in gene-rich regions within an individual genome and the “patchy” distribution among different strains point to the mobile elements as major generators of verticillium intra- and inter-specific genomic variation. we report also of their distribution in other vd and va isolates from various geographic origins. to date, no sexual stage has been identified in either microorganism suggesting that somatic mutation is a major force in generating genetic diversity. revealed that non-random insertions of transposable elements have contributed to the generation of four lineage-specific regions in vdls. transposable elementsverticillium spp.retrotransposonsdna transposonsrepeat-induced point mutation te domesticationgenome evolution we present here a detailed analysis of class i retrotransposons and class ii “cut-and-paste” dna elements detected in the sequenced verticillium genomes. while copia-, gypsy- and tc1/mariner-like transposons were prominent, a large variation in presence of the other types of mobile elements was detected in the other verticillium spp. noticeably, we found evidences of repeat-induced point mutation only in some of the gypsy retroelements. copia-, gypsy- and tc1/mariner-like transposons are the most wide-spread tes in the phytopathogens v. dahliae and v. albo-atrum. some of these elements have undergone diversification and subsequent selective amplification after introgression into the fungal genome. verticillium dahliae and verticillium albo-atrum are cosmopolitan soil fungi causing very disruptive vascular diseases on a wide range of crop plants.
 verticillium dahliae and verticillium albo-atrum are cosmopolitan soil fungi causing very disruptive vascular diseases on a wide range of crop plants. to date, no sexual stage has been identified in either microorganism suggesting that somatic mutation is a major force in generating genetic diversity. whole genome comparative analysis of the recently sequenced strains vdls. and vams. revealed that non-random insertions of transposable elements have contributed to the generation of four lineage-specific regions in vdls.  we present here a detailed analysis of class i retrotransposons and class ii “cut-and-paste” dna elements detected in the sequenced verticillium genomes. we report also of their distribution in other vd and va isolates from various geographic origins. in vdls. we identified and characterized complete retrotransposons of the gypsy-, copia- and line-like types, as well as full-length elements of the “cut-and-paste” superfamilies tc1/mariner, activator and mutator. while copia and tc1/mariner were present in multiple identical copies, activator and mutator sequences were highly divergent. most elements comprised complete orfs, had matching ests and showed active transcription in response to stress treatment. noticeably, we found evidences of repeat-induced point mutation only in some of the gypsy retroelements. while copia-, gypsy- and tc1/mariner-like transposons were prominent, a large variation in presence of the other types of mobile elements was detected in the other verticillium spp. strains surveyed. in particular, neither complete nor defective “cut-and-paste” tes were found in vams.  copia-, gypsy- and tc1/mariner-like transposons are the most wide-spread tes in the phytopathogens v. dahliae and v. albo-atrum. in vdls. we identified several retroelements and “cut-and-paste” transposons still potentially active. some of these elements have undergone diversification and subsequent selective amplification after introgression into the fungal genome. others, such as the ripped copias, have been potentially acquired by horizontal transfer. the observed biased te insertion in gene-rich regions within an individual genome and the “patchy” distribution among different strains point to the mobile elements as major generators of verticillium intra- and inter-specific genomic variation. transposable elementsverticillium spp.retrotransposonsdna transposonsrepeat-induced point mutation te domesticationgenome evolution 