ORIGINAL PAPER

Peter W. Inglis Æ Daniel J. Rigden

Luciane V. Mello Æ Edward J. Louis

M. Cleria. Valadares-Inglis

Monomorphic subtelomeric DNA in the filamentous fungus,Metarhizium anisopliae, contains a RecQ helicase-like gene

Received: 27 August 2004 / Accepted: 19 April 2005 / Published online: 2 June 2005� Springer-Verlag 2005

Abstract In most filamentous fungi, telomere-associatedsequences (TASs) are polymorphic, and the presence ofrestriction fragment length polymorphisms (RFLPs)may permit the number of chromosome ends to beestimated from the number of telomeric bands obtainedby restriction digestion. Here, we describe strains ofMetarhizium, Gliocladium and Paecilomyces species inwhich only one or a few telomeric bands of unequalintensity are detectable by Southern hybridization,indicating that interchromosomal TAS exchange occurs.We also studied an anomalous strain of Metarhiziumanisopliae, which produces polymorphic telomeric bandslarger than 8 kb upon digestion of genomic DNA withXhoI. In this case, the first XhoI site in from the chro-mosome end must lie beyond the presumed monomor-phic region. Cloned telomeres from this strain comprise18–26 TTAGGG repeats, followed at the internal end ofthe telomere tract by five repeats of the telomere-likesequence TAAACGCTGG. An 8.1-kb TAS clone alsocontains a gene for a RecQ-like helicase, designatedTAH1, suggesting that this TAS is analogous to the Y¢elements in yeast and the subtelomeric helicase ORFs ofUstilago maydis (UTASRecQ) and Magnaporthe grisea(TLH1). The TAS in the anomalous strain of M. ani-sopliae, however, appears distinct from these in that it isfound at most telomeres and its predicted proteinproduct possesses a significantly longer N-terminal re-gion in comparison to the M. grisea and U. maydis he-licases. Hybridization analyses showed that TAH1

homologues are present in all other anomalous M. ani-sopliae strains studied, as well as in some other poly-morphic strains, where the recQ-like gene also appearsto be telomere-associated.

Keywords Fungi Æ RecQ helicase ÆTelomere associated sequence Æ Recombination

Introduction

Filamentous fungi are of interest as biocontrol agentsdue their ability to parasitize and cause disease in manyspecies of insects, weeds and fungal plant pathogens.Entomopathogenic fungi, particularly species of Meta-rhizium, Beauveria and Paecilomyces,are used to controla variety of insects in agriculture (Prior 1992; Magalhaes1997; Langewald et al. 1999; Inglis et al. 2001a), whileother fungi, such as Gliocladium species, are in use tocontrol important diseases in a variety of crops (Wraightet al. 2001). The success of a commercial biopesticidedepends on the genetic stability of the organism duringinoculum production and storage (Moore et al. 1993).Furthermore, the pathogenicity of isolates of somespecies has been reported to decline upon repeatedsubculturing in vitro (Fargues and Roberts 1983). Phe-notypic degeneration has also been observed to occurduring sector formation in some species, such as Meta-rhizium anisopliae, and is frequently accompanied bychanges in the ability to produce secondary metabolitesand enzymes (Ryan et al. 2002). However, little is knownabout the mechanisms associated with genetic variationin these organisms.

We previously studied the genetic variability of agroup of M. anisopliae var. acridum (= M. flavoviride)isolates by restriction fragment length polymorphism(RFLP) using a telomeric probe (Inglis et al. 1999), andfound the telomere-associated region to be highly poly-morphic. We have also used this technique to investigategenetic variability in our collection of entomogenous

Communicated by E. di Mauro

P. W. Inglis (&) Æ D. J. Rigden Æ L. V. MelloM. C. Valadares-InglisEmbrapa Recursos Geneticos e Biotecnologia,Parque Estacao Biologica, Final Av. W/5 Norte,Brasılia-DF, CEP 70770900, C.P., 02372, BrazilE-mail: peteringlis@onetel.comTel.: +55-61-4484784Fax: +55-61-4484673

E. J. LouisDepartment of Genetics, University of Leicester,Leicester, LE7 1RH, UK

Mol Gen Genomics (2005) 274: 79–90DOI 10.1007/s00438-005-1154-5

M. anisopliae var. ansiopliae and Paecilomyces fumos-oroseus species, and in isolates of Gliocladium that showantifungal activity. These fungi are all members of theascomycete order Hypocreales. Among these, we iden-tified strains with unusually homogeneous telomere-associated regions that yield a highly intense band andone or two weakly hybridizing fragments on Southernblots probed with a telomeric probe upon digestion withvarious restriction enzymes. However, most Metarhizi-um strains produce 12–14 equally intense telomericbands, indicating the presence of six or seven chromo-somes in this fungus, as confirmed by electrophoretickaryotyping (Valadares-Inglis and Peberdy 1998). Thephenomenon of monomorphic telomere-associatedDNA was previously noted in some strains of Botrytiscinerea, but most strains of this species also possesshighly polymorphic telomere-associated DNA (Leviset al. 1997).

The lack of polymorphism in these telomere-associ-ated sequences (TASs) could be explained by eithermultiple endogenous recombination events betweenchromosome ends, which leads to homogenization inthese strains, or by the activity of a transposable elementthat preferentially inserts at a telomeric or subtelomerictarget site. Thus, a degenerate LTR transposon, desig-nated Pogo, has been identified at the VR telomere ofNeurospora crassa (Schectman 1990). However, otherputative copies of this element, identified by hybridiza-tion, were found only at several other non-telomericsites. In Drosophila melanogaster, telomeres are natu-rally maintained by transposition and tandem duplica-tion of specialized non-long terminal repeat (non-LTR)retrotransposons, including the LINE-like HeT-A whichtransposes to broken chromosome ends (Danilevskayaet al. 1997), and TART (telomere-associated retro-transposon) which also transposes and duplicates atnatural chromosome ends (Levis et al. 1993). Other non-LTR retrotransposons, such as TRAS1 (Okazaki et al.1995) and SART1 (Takahashi et al. 1997), containgag- and pol-like ORFs and a 3¢ poly(A) tract, and areinserted at specific sites in the telomeric repeats in thesilkworm Bombyx mori.

In Saccharomyces cerevisiae, a mosaic of differentrepeats, varying from end to end and differing amongstrains, is present in the telomere-associated sequences,and these may or not contain ORFs. The subtelomericDNA of about half the chromosome ends in S. cerevisiaecontains the so-called Y¢ element, in long and shortforms; the function and origin of this element is uncer-tain (see review by Louis 1995). However, amplificationof the Y¢ element is part of a mechanism, which enablesthe cell to survive in the absence of telomerase activity(Lundblad and Blackburn 1993; Louis 1995; Yamadaet al. 1998).

Due to the potential implications of telomere-asso-ciated DNA rearrangements for genome variability andstability in filamentous fungi, we cloned and character-ized the monomorphic telomere-associated DNA of theM. anisopliae strain CG34. Its structure may help toexplain the mechanism by which such telomere-associ-ated DNAs are duplicated or transferred among chro-mosomes in such isolates.

Materials and methods

Strains

Ten strains of P. fumosoroseus, 14 strains of M. ani-sopliae var. anisopliae and 19 strains of Gliocladium sp.were used in our analysis of subtelomeric polymor-phism. The M. anisopliae and P. fumosoroseus strains(see Tables 1 and 2 for details of their origins) wereobtained from the collection of entomopathogenic fungimaintained by Embrapa Genetic Resources and Bio-technology (CENARGEN). The Gliocladium sp. strains,all of which were distinct soil isolates collected in Brazil,were obtained from the Embrapa Grape and WineCulture Collection (kindly provided by Dr. R.M. San-hueza). Isolates were maintained as conidial stocksstored in liquid nitrogen and were cultivated onpotato dextrose agar (Difco) for production of conidialinoculum. M. anisopliae var. anisopliae strain CG34,used for TAS cloning, was originally isolated from

Table 1 Origin of M. anisopliae var. anisopliae strains

No. Strain Date Geographical origin Host/substrate

1 CG29 1988 Manaus, AM, Brazil Dois flavopicta (Homoptera: Cercopidae)2 CG32 1988 Bahia, Brazil Mahanarva posticata (Homoptera: Cercopidae)3 CG34 1988 Manaus, AM, Brazil Conotrachelus sp. (Coleoptera: Curculionidae)4 CG38 1992 Porto Velho/RO, Brazil Coleoptera: Scarabaeidae5 CG100 1988 Brasilia, DF, Brasil Coleoptera: Scarabaeidae6 CG210 1982 Goiania, GO, Brazil Ceratoma arcuata (Coleoptera: Chrysomelidae)7 CG236 1982 Goiania, GO, Brazil Coleomegilla maculata (Coleoptera: Coccinelidae)8 CG292 1990 Adelaide, Australia Aphodius tasmaniae (Coleoptera: Scarabaeidae)9 CG322 1990 France Melolontha melonontha (Coleoptera: Scarabaeidae)10 CG324 1985 Quebec, Canada Phyllophaga anxia, Coleoptera: Scarabaeidae11 CG374 1990 South Pacific Islands Coleoptera: Scarabaeidae12 CG390 1994 New Zealand Coleoptera: Scarabaeidae13 CG490 1987 New Zealand Conoderus sp. (Coleoptera: Elateridae)14 CG491 1983 Londrina, PR, Brazil Dois sp.

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Conotrachelus sp. (Coleoptera: Curculionidae) in Man-aus (Amazonas State, Brazil) in 1988.

Telomere fingerprints

Extraction of fungal genomic DNA from liquid-grownmycelium, and analysis of telomeric RFLPs weredescribed previously (Inglis et al. 1999). In order to mapthe extent of the element associated with the monomor-phic telomeres in strain CG34, genomic DNA was di-gested with a range of restriction enzymes, and Southernblots were probed with the telomeric sequence fromFusarium oxysporum [TTAGGG]18 (Powell and Kistler1990).

Bal31 digestion of genomic DNA from M. anisopliaestrains CG34 and CG100

The terminal nature of the telomeric bands revealedusing the telomeric probe [TTAGGG]18 in the anoma-lous strain CG34 and the normal strain CG100 wasverified by partial digestion of genomic DNA with Bal31nuclease, using a modification of the method of Weiss-Schneeweiss et al. (2004). Briefly, 1 lg of genomic DNAwas digested with 1 U of Bal31 nuclease (New EnglandBiolabs) in the buffer recommended by the manufac-turer, for 0, 15, 30 and 60 min. Reactions were stoppedby phenol–chloroform extraction, and the samples weredigested to completion with EcoRI. A Southern blot wasthen probed with the telomeric sequence [TTAGGG]18.

Preparation and screening of a clone libraryenriched for terminal sequences from M. anisopliaestrain CG34

End-repair of telomeres was conducted using a modifi-cation of the procedure of Bhattacharyya and Blackburn(1997). CG34 genomic DNA (10 lg) was treated with5 U of Vent DNA polymerase (New England Biolabs) inthe recommended buffer containing 1 mM dNTPs at70�C for 1 h. The now blunt-ended genomic DNAfragments were ligated, using T4 DNA ligase, to EcoRV-

digested pBluescript II SK+ plasmid (1 lg; Stratagene)at 4�C overnight. The ligation mixture was then digestedto completion with XhoI, extracted with phenol–chloro-form and religated with T4 DNA ligase. This ligationmixture, enriched for fragments containing repairedterminal DNA, was then used to transform competentEscherichia coli XL10-Gold cells (Stratagene). Trans-formed cells were plated on LB agar supplemented withampicillin, IPTG and X-gal. Plates were then incubatedat 28�C for 36 h. This low incubation temperature wasfound to be essential for recovery of clones containingM. anisopliae telomeric repeats, presumably due toinstability of this repetitive sequence in E. coli at 37�C.Colony lifts were prepared on Genescreen plus mem-branes (Du Pont) according to standard procedures. Thetelomere-enriched XhoI library was then screened usingthe a�32 P[dCTP]-labeled F. oxysporum telomeric probe.Colonies that gave positive hybridization signals wereanalysed by restriction mapping. One clone whoserestriction map was compatible with Southern hybrid-ization data for the uncloned TAS was then selected forfurther study, and designated pMAT3A1. Another fivechromosome end clones were also partially sequenced,using the M13 Reverse sequencing primer, which coversthe terminal DNA ligated to the EcoRV half-site of thevector.

Sequence analysis of the telomeric clone pMAT3A1

Nested deletion sets for both strands of pMAT3A1 wereprepared using the Exonuclease III digestion technique(Henikoff 1987). Clones were sequenced by the chain-termination method using the Thermo-Sequenase radio-labelled terminator cycle sequencing kit (Amersham) anda�33 P-labelled ddNTPs. Initial analysis of the pMA-T3A1 sequence revealed the presence of a large ORF. Thesequence of the predicted product was then subjected tohomology analysis. Related sequences were found usingFASTA (Pearson and Lipman 1988) or BLAST (Altschulet al. 1997) searches of the Swissprot and GenBankdatabases. Multiple alignments and similarity assess-ments of full-length and partial sequences were carriedout using ClustalX, and a neighbor-joining tree incor-porating bootstrapped data was drawn with NJPLOT.

Table 2 Origin of P. fumosoroseus strains

No. Strain Date Geographical origin Host/substrate

1 CG35 1991 Brasilia, DF, Brasil Soil2 CG123 1987 Manaus, AM, Brazil Spaethiella sp. (Coleoptera: Chrysomelidae)3 CG170 1989 Apopka, FL, USA Pseudococcus sp. Homoptera: Pseudococcidae4 CG204 1990 Mexico Bemisia sp. (Homoptera: Aleyrodidae)5 CG323 1990 Japan Bombyx mori (Lepidoptera: Bombycidae)6 CG325 1985 Philippines Nilaparvata lugens (Homoptera: Delphacidae)7 CG397 1992 Florida, USA Bemisia tabaci (Homoptera: Aleyrodidae)8 CG404 1992 Kathmandu, Nepal Bemisia tabaci (Homoptera: Aleyrodidae9 CG186 1991 Pelotas, RS, Brazil Soil10 CG260 1991 Pelotas, RS, Brazil Anastrepha sp. (Diptera: Tephritidae)

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Software packages were used with default settings whereappropriate. The sequence of the insert in clone pMA-T3A1 has been deposited with the EMBL/Genbank DataLibraries under the accession no. AF291909.

Southern analysis of fungal DNAs with the recQ-likesequence from clone pMAT3A1

Southern blots of M. anisopliae, P. fumosoroseus andGliocladium spp. DNAs that had previously been anal-ysed with the telomeric repeat probe were re-probedwith the EcoRV– BamHI fragment from pMAT3A1,which includes the bulk of the reqQ-like ORF fromM. anisopliae strain CG34. Hybridization was carriedout at moderate stringency (60�C in 1· Church andGilbert buffer) and was followed by extended exposureto X-ray film to detect weakly hybridizing bands.

Northern analysis of the recQ-like transcriptin M. anisopliae strain CG34

M. anisopliae strain CG34 was grown in 100 ml ofpotato dextrose broth (Difco) in a shake flask at 28�C

for 72 h. Harvested fungal mycelium was ground to apowder in liquid nitrogen, and total RNA was extractedusing an RNeasy kit (Qiagen). A northern blot bearingthe total RNA was then probed with the EcoRV–BamHI fragment from pMAT3A1.

Results

Monomorphic telomere-associated DNA in strainsof Gliocladium sp., P. fumosoroseus and M. anisopliae

Genomic DNA fingerprinting analysis of M. anisopliae,P. fumosoroseus and Gliocladium sp. using the telomericprobe [TTAGGG]18 revealed anomalously strong bandsin some strains of these fungi (Fig. 1), in contrast to thehighly polymorphic patterns observed in most strains.Among 13 strains of P. fumosoroseus analysed, only oneproduced a monomorphic band when digested withHindIII, EcoRI or PstI, while in Gliocladium sp. mono-morphic telomere-associated DNA was observed in 21%of the strains analysed. Several different M. anisopliaeisolates were also found to display monomorphic orotherwise anomalous telomere fingerprints.

InM. anisopliae strains possessing polymorphic TASs,the number of telomeric bands observed varied widely, upto a maximum of 18 bands in strain CG322, which wouldcorrespond to a complement of nine chromosomes.Electrophoretic karyotype data are available for strainCG34. Like two other Brazilian M. anisopliae strains,

Fig. 1 Analysis of telomeric RFLP. Genomic DNAs from non-selected monosporic strains of Gliocladium sp. (digested with PstI),P. fumosoroseus (digested with PstI) and M. anisopliae (digestedwith EcoRI) were probed with the telomeric sequence[TTAGGG]18. The strains marked with arrowheads have mono-morphic or near-monomorphic TASs

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CG34 showed seven chromosomes; in a fourth isolateeight chromosomes were resolved (Valadares-Inglis andPeberdy 1998). Shimizu et al. (1992) have reported sevenchromosomes in a group of M. anisopliae isolates fromJapan. In strains with polymorphic subtelomeric DNA,the correlation between the number of bands detectedwith a telomeric probe and the chromosome number ar-gues against the presence of significant tracts of telomericrepeats at non-telomeric loci in these strains. In M. ani-sopliae strain CG34, which gives anomalous telomericfingerprints, and strain CG100, which yields normaltelomeric fingerprints, Bal31 nuclease analysis confirmedthat the bands that hybridized with the telomeric probewere exclusively terminal (Fig. 2).

The telomere-associated DNA of strain CG34 wasthen mapped in greater detail using different restrictionenzymes (Fig. 3). The majority of enzymes produced asimilar restriction pattern to EcoRI, yielding a singleintense telomeric band with one or two weakerbands. However, NotI, SmaI, SpeI and XhoI produced

polymorphic telomeric profiles consisting of high-molecular-weight fragments, indicating that the recog-nition sites for these enzymes lie beyond the regionpresumed to be common to all chromosomes. Themonomorphic bands obtained with the other enzymesranged in size from 450 bp (SacI) to approximately7,000 bp (ApaI). SacI and XbaI digests also producedfive and four weaker bands, respectively, of low molec-ular weight, in addition to the intensely hybridizingband, suggesting that certain copies of the duplicatedelement contain polymorphisms.

Cloning and sequence analysisof the telomere-associated sequencefrom M. anisopliae strain CG34

XhoI was chosen as the restriction enzyme for cloningpurposes, since it was found to produce well-resolved,apparently fully polymorphic telomeric bands withmolecular weights greater than 8.0 kb. Following enzy-matic repair of M. anisopliae CG34 genomic DNA ends,a terminal DNA enriched library was constructed inpBluescript II SK+, which was used to transform

Fig. 2 Bal31 nuclease analysis of M. anisopliae strains CG34 andCG100. Genomic DNA (1 lg/lane) was digested for the timesindicated (in min) above the lanes. DNA was then digested tocompletion with EcoRI and a Southern blot was probed with[TTAGGG]18

Fig. 3 Restriction mapping of monomorphic telomere-associatedDNA in theM. anisopliae strain CG34. Various restriction enzymeswere used to digest the genomic DNA and Southern blots wereprobed with 32 P-labelled [TTAGGG]18

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competent E. coli XL10-Gold cells (Stratagene). Sixclones that hybridized to the [TTAGGG]18 probe wereidentified, and these were digested with restriction en-zymes in order to identify those that matched the map-ping data previously obtained. One of three matchingclones was selected for full sequence analysis. This cloneof M. anisopliae telomeric and TAS DNA, designatedpMAT3A1 (Fig. 4), was found to contain an insert ofapproximately 8.0 kb.

The telomere repeats ligated to the EcoRV half-site offour of the terminal DNA clones, including pMAT3A1,was found to consist of variable numbers of the hex-anucleotide tandem repeat TTAGGG. This is identicalto the telomere repeat units found in humans and othervertebrates (Meyne et al. 1989), in the slime mouldPhysarum (Forney et al. 1987), in the protozoan Try-panosoma (Blackburn and Challoner 1984) and in thefilamentous fungi Neurospora crassa (Schectman 1990),Ustilago maydis (Guzman and Sanchez 1994) andAspergillus nidulans (Bhattacharyya and Blackburn1997). In other clones containing M. anisopliae telo-meres, pMAT1B1 had 20 TTAGGG repeats, pMAT3A1had 18 repeats, pMAT7C1 had 23 repeats and pMA-T10A4 had 26 repeats, which equates to a telomerelength of between 108 and 156 bp. Immediately adjacentto the TTAGGG repeats in all clones, including clonepMAT1B1 (which does not match the restriction map ofthe duplicated element) are five perfect repeats of thesequence TAAACGCTGG, which appear to form partof the telomeric array. This telomere-like sequenceappears to be unique to M. anisopliae and is not foundadjacent to the telomeres of other filamentous fungi forwhich sequence data are available.

No LTRs or target site duplications that would becharacteristic of LTR retrotransposon insertion wereidentified at the centromere-proximal end of the pMA-T3A1 clone (McHale et al. 1992). However, a large ORFof 4,553 bp was identified at the centromere-proximalend of pMAT3A1, 2.6 kb from the first [TTAGGG]

repeat unit (Fig. 4). BLAST (Altschul et al. 1997) sear-ches using the amino acid sequence deduced from thelarge ORF as the query revealed significant similaritybetween the M. anisopliae telomere-associated ORF andknown genes for RecQ family helicases. The best hitswere two highly similar telomere-associated genes forRecQ-like helicases from the fungus U. maydis (Swiss-prot identifiers O13399 and O13400; Sanchez-Alonsoand Guzman 1998), which gave E values of around10�33 with 34% sequence identity in a 401-residueoverlap. These matches were followed by helicases giv-ing E values of 10�14 and above with typically 26%identity over stretches of about 480 residues (data notshown). These results enabled the assignment of theproduct of the pMAT3A1 ORF as a member of theDEAD/DExH family of helicases (for a review, see de laCruz et al. 1999), and we named this protein TAH1 (forM. anisopliae telomere-associated helicase 1). No sig-nificant similarity was found between the M. anisopliaeTAS and a class of transposable element, the helitrons,recently described in fungi including Phanarochaetechrysosporium (Poulter et al. 2003). Subsequent detailedsequence comparisons were made with RecQ-familyhelicases from filamentous and other fungi.

FASTA alignments (Pearson and Lipman 1988)extending over the majority of the conserved helicasecore of TAH1 showed that the Metarhizium sequenceagrees well with consensus helicase motifs (Fig. 5). TheP-loop and DExH sequences (motifs I and II, Fig. 5) areinvolved in binding ATP, while motif III (TGTLP in M.anisopliae) forms a flexible linker between the first twodomains of the helicase structure. In motif VI the twoArg residues proposed to bind ATP upon domain clo-sure are conserved. Phylogenetic analysis was also car-ried out on the conserved helicase domain of thededuced M. anisopliae TAH1 sequence in comparisonwith other selected helicases (Fig. 6). The tree topology,which was generally well supported by the results of1,000 bootstrap replicates, showed that the subtelomer-ically encoded helicases of the filamentous fungiM. anisopliae, U. maydis and M. grisea formed a diversecluster separate from the other helicases analysed, withthe Y¢ helicase from yeast being a highly diverged out-lier. The other non-subtelomeric RecQ-family helicases,

Fig. 4 Organization of the telomeric clone pMAT3A1. Mappositions are derived from sequence data, but agree with theSouthern hybridization data shown in Fig. 2. The full-length DNAsequence of the pMAT3A1 clone has been deposited in theGenBank/EMBL databanks under the accession no. AF291909

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including the yeast SGS1 and human WRN and BLMhelicases, formed a distinct clade of higher overall sim-ilarity than that encompassing the subtelomeric heli-cases. Cluster analysis of the complete amino acidsequences of these helicases produced a tree topologyclosely resembling that recovered on the basis of thehelicase domains alone (data not shown). This is likelyto be related to the degree of divergence of thesesequences outside of the conserved helicase core. Fur-thermore, the relative position of the helicase domain inthe proteins was also variable, being biased eithertowards the central region of the proteins in M. anisop-liae, or towards the N-terminal end of the proteins in theRecQ helicases from U. maydis and M. grisea (Fig. 7).

Southern and northern hybridization analysisof the TAH1 sequence from M. anisopliaestrain CG34

Significant cross-hybridization was detected between theEcoRV– BamHI fragment from pMAT3A1, which rep-resents the bulk of the TAH1 gene from M. anisopliaestrain CG34, and the DNA of several other M. anisop-liae strains digested with EcoRI. Strains that cross-hybridized significantly with the probe included all thosepossessing anomalous telomere fingerprints, namely,CG29, CG38, CG210 and CG292 (Fig. 8). In strainspossessing normal, polymorphic TASs, cross-hybridiza-tion with the probe was either extremely weak or absent.Exceptions to this were the completely polymorphicstrains CG490 and CG491, in which strongly hybridiz-ing signals were obtained. In several cases, some of thecross-hybridizing bands appeared to have the samemolecular weights as a particular telomeric band. Thisholds for strain CG210, where two (the largest) of thethree bands detected exactly matched bands thathybridized with the [TTAGGG]18 probe, and for strainsCG490 and CG491, where the TAH1-hybridizing bandsalso appeared to be identical in size to those thathybridized to the telomeric probe. Under the conditionsutilized, no significant hybridization signals were de-

Fig. 5 Multiple alignment (ClustalX) of the core domains ofselected helicases of the RecQ family. The shaded areas indicatethe conserved helicase motifs proposed by Gorbalenya et al. (1989).The genes aligned were: RECQ: E. coli recQ helicase gene;DMBLM: Drosophila melanogaster recQ helicase homolog; BLM:Homo sapiens Bloom Syndrome gene; MUSN: Aspergillus nidulansRecQ helicase; QDE3: Neurospora crassa QDE3 gene (similar toRecQ helicase); SGS1: Saccharomyces cerevisiae DNA helicasegene; WRN: H. sapiens Werner Syndrome gene; UTAS: Ustilagomaydis clone UT5 telomere-associated recQ-like helicase (UTAS-recQ) gene; TLH1: Magnaporthe grisea telomere-linked helicase 1gene; TAH1: Metarhizium anisopliae telomere-associated helicasegene (this study)

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tected with the M. anisopliae RecQ helicase probe ingenomic DNAs of P. fumosoroseus or Gliocladium spp.Furthermore, no TAH1 transcript could be detected onnorthern blots bearing RNA from M. anisopliae strainCG34 grown under normal conditions.

Discussion

We have described here our observations on polymor-phism in subtelomeric sequences in strains of the fila-mentous fungi Gliocladium sp., P. fumosoroseus andM. anisopliae. Telomeric probes have previously beenused for DNA fingerprinting of filamentous fungi suchas U. maydis (Sanchez-Alonso et al. 1996), Metarhiziumsp. (Inglis et al. 1999), Nomuraea rileyi (Boucias et al.2000), Cercospora caricis (Inglis et al. 2001b) andBeauveria sp. (Viaud et al. 1998). On the basis of thesestudies, a high level of subtelomeric polymorphism isexpected. However, we observed that strains that yieldanomalously low numbers of telomeric bands appear to

occur relatively frequently in our collection of filamen-tous fungi. A TAS clone, pMAT3A1, derived from theanomalous M. anisopliae strain CG34 contains a gene(TAH1) for a RecQ-like helicase, and the restriction mapof this clone matches the pattern expected from genomictelomeric fingerprinting in this strain. It is likely, there-fore, that the anomalous pattern of telomeric fragmentshas arisen by the acquisition of a TAS containing thisTAH1 gene by most of the telomeres. Apparently in-terchromosomal recombination has placed this sequencein a fixed position relative to the telomere repeats(Fig. 9). Most strains that give anomalous telomericfingerprints displayed a few weak bands in addition toone highly intense telomeric band, suggesting that theevent responsible for the apparent terminal inter-chromosomal duplications in these strains either did notinvolve all chromosome ends, or that the sequencebecame unstable at some point. However, strains CG292(Fig. 1, M. anisopliae, No. 8), CG323 (Fig. 1, P. fu-mosoroseus, No. 5), 3GDO1, 10GDO2, GSJ1 and GFO3(Fig. 1, Gliocladium sp. Nos. 4, 9, 12 and 15) showed a

Fig. 6 Unrooted neighbor-joining tree (Saitou and Nei1987) based on helicase domainsequences of selected RecQ-family helicases from Fig. 5.Percent divergences werecalculated using ClustalX andare indicated by the scale bar.The results of 1,000 bootstraptrials of the branch data areindicated on the tree nodes. Thetree was drawn using NJPlot

Fig. 7 Aligned maps of selectedRecQ-family helicases showingthe relative lengths of theproteins, and the locations ofthe conserved helicase domains.Sequences include the aminoacid sequence deduced from theM. anisopliae TAH1 ORFpresent in the clone pMAT3A1.Adapted from Bjergbaek et al.(2002)

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single intense telomeric band, suggesting that in thesecases all chromosomes were affected by either anamplification event targeted to the telomere or subtelo-mere, or by extensive recombination resulting in thehomogenization of the chromosome ends. This lack ofTAS polymorphism, at least within the DNA size rangethat is resolved by standard agarose gel electrophoresis,appears to be a common phenomenon in these strains offilamentous fungi. In the cases of P. fumosoroseus andM. anisopliae, we could not correlate the presence ofmonomorphic TASs with the host of origin or the geo-graphic source of the fungal isolates examined. All theGliocladium sp. isolates used in this study originatedfrom Brazilian soils and were originally selected on thebasis of their ability to suppress other moulds. More-over, possession of a monomorphic TAS did notcorrelate with groups recognized on the basis of RAPDPCR typing (data not shown). Furthermore, thetelomeric fingerprints of the remainder of the Gliocladi-um isolates with polymorphic TAS showed the group tohave generally low genetic similarity. This suggests that

the acquisition of monomorphic TAS in these filamen-tous fungi is a random event that has occurred inde-pendently in different isolates.

Although M. anisopliae strain CG34 possesses amonomorphic TAS, the [TTAGGG]n telomere repeatarrays appear normal. The telomere lengths in CG34 areof a similar order to those seen in A. nidulans (�100 bp;Bhattacharyya and Blackburn 1997) and in the VRtelomere of N. crassa (162 bp; Schectman 1990). Thevariation observed in telomere length in the partiallysequenced M. anisopliae TAS clones is unlikely to be anartifact of enzymatic processing with the proofreadingDNA polymerase used since, in the presence of excessdNTPs, loss of telomeric DNA should be minimal(Zakian 1989). Furthermore, telomere-repeat arrays areknown to show heterogeneity in length even whenderived from a particular chromosome (Zakian 1989).Little systematic information is available on telomerelength in filamentous fungi, since telomeres are usuallypoorly represented in the libraries used for genomesequencing projects. For example, no clones hybridizingto a Neurospora telomeric sequence (Schectman 1990)were found in a N. crassa cosmid library of 12,000 clones(at least nine genome equivalents; Kelkar et al. 2001).

Among the filamentous fungi, genes for RecQ-likehelicases have been found in the subtelomeric DNAs ofU. maydis (UTASrecQ; Sanchez-Alonso and Guzman1998) and Magnaporthe grisea (TLH1, Gao et al. 2002).In the U. maydis strain FB2 a2b2, the helicase gene wasfound on a clone containing the subtelomeric repeat,UTASa. This repeat hybridized to 15 chromosomes inelectrophoretic karyotypes of the source strain, buthybridized to only two chromosomes in a second strain,I2. The number of individual chromosome endspossessing a copy of UTASrecQ has not been reported.In two isolates of M. grisea from rice, a RecQ-likehelicase gene (designated TLH1,) which resemblesUTASrecQ from U. maydis, was found within 15 kb ofat least seven and four of the 14 chromosome ends,respectively (Gao et al. 2002). TLH1 was detected byhybridization, with a copy number between two and five,in more than 100 M. grisea strains isolated from riceand collected in Korea in 1999. This prevalence was

Fig. 9 Presumed mechanism ofchromosome endhomogenization. A single TAS(with an embedded helicasegene) is used as a template forrecombination-dependenttransfer of TAS to otherchromosome ends, resulting inthe reduction or elimination ofsubtelomeric restrictionfragment polymorphism. Theletters above the bars indicatearbitrary unique restriction sitesand the circles representtelomere repeats

Fig. 8 Southern hybridization analysis of M. anisopliae strains(labelled 1–14 as in Table 1). Genomic DNA was digested withEcoRI. The EcoRV– BamHI fragment of pMAT3A1, containingthe bulk of the M. anisopliae TAH1 gene, was used as the probe

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compatible with the fact that the strains exhibited over90% identity in RFLPs detectable with a repetitiveDNA probe (Gao et al. 2002). However, the TLH1 genefamily could not be detected by hybridization in mostisolates recovered from host plants other than rice. Incontrast, cross-hybridization with the TAH1 probe fromCG34 reveals that the gene shows no correlation withhost origin, as homologous sequences are found in M.anisopliae strains collected from both the insect ordersHomoptera and Coleoptera, as well as from differentfamilies within the Coleoptera.

At least in M. anisopliae, the appearance of anoma-lous or monomorphic TAS was strongly associated withthe possession of a TAH1 homologue. The strength ofthe hybridization signals obtained was highly variable,indicating significant sequence divergence in this genebetween isolates. This variability may even apply tomultiple copies of the gene in the same isolate. InU. maydis, the sequence of the helicase gene from asecond clone of UTASa from strain FB2 a2b2 differedfrom the first by 10% (Sanchez-Alonso and Guzman1998). The strong divergences in RecQ-like helicase se-quences found in these fungi probably account for thelack of significant hybridization signals with DNA fromGliocladium spp. and P. fumosoroseus. Interestingly, itseems that telomere-associated TAH1 homologues in thepolymorphic M. anisopliae strains CG490 and CG491are closely related to the gene from CG34, despite thedifferent geographical and host origins of these strains.

The role of the RecQ helicases in the amplificationand homogenization of subtelomeric DNA of M. ani-sopliae, U. maydis and M. grisea is currently unclear,although it may be related in some way to the mainte-nance of genome stability. One of the best-understoodRecQ-type helicases is SGS1 from S. cerevisiae, which isrequired for faithful chromosome segregation (Wattet al. 1995, 1996). Yeast sgs1 mutants are also subject topremature senescence (Sinclair et al. 1997). Deleteriousmutations in the human RecQ-genes involved inBloom’s disease (BLM; Ellis et al. 1995) and Werner’sdisease (WRN; Yu et al. 1996) give rise to prematureageing phenotypes and a predisposition to cancer, whichare reflected in genetic instability at the cellular level.

Telomere-targeted interchromosomal duplication ofan element encoding a RecQ-like helicase in M. anisop-liae strain CG34 has strong parallels with the Y¢ ele-ments of yeast, which are present in single or multipletandem copies in subtelomeric DNA (Louis and Haber1992). In the sequenced strain of S. cerevisiae, S288C, Y¢elements are found at 17 out of 32 telomeres (Louis1995). In comparison, our restriction mapping datasuggest that the TAH1-containing TAS in M. anisopliaestrain CG34 is likely to be present at 12 of the 14chromosome ends. The yeast Y¢ element possesses anORF for a product that shows 27% identity to helicases(although not closely related to RecQ helicases), andexists in two forms, long and short, which differ byseveral insertions or deletions within this region (Louisand Haber 1992). Y¢ elements are highly conserved,

showing only 0.3–1.13% sequence divergence within astrain and 1.15–1.75% divergence between strains, con-trasting with 8–18% divergence among adjacent subtel-omeric repeats. Yeast cells in which telomerase has beeninactivated are known to senesce due to gradual erosionof telomeres. A small proportion of the senescent cellsare, however, able to survive and multiply by activationof a telomerase-independent mechanism of telomeremaintenance (Lundblad and Blackburn 1993). Thesesurvivors can be divided into two types: type I survivorshave high levels of amplification of Y¢ elements in tan-dem arrays, whereas type II survivors have very longtracts of [TG1-3] telomere repeats. Although the struc-ture and distribution of Y¢ suggests an origin as a mobileelement, activation and the development of both types oftelomerase-deficient survivors is dependent on recombi-nation and requires RAD52 (Lundblad and Blackburn1993) and either RAD50 or RAD51 (Teng and Zakian1999). Furthermore, production of type II survivors hasbeen shown to be dependent on SGS1 (Cohen and Sin-clair 2001; Huang et al. 2001).

We were not able to detect a TAH1 transcript bynorthern blotting of RNA from M. anisopliae strainCG34, grown under normal conditions. Likewise,northern analysis failed to detect transcripts from anM. grisea strain that has an amplified subtelomericTLH1 gene, when grown under a range of conditionsincluding carbon and nitrogen-source starvation, heatand cold shocks and exposure to mutagenic agents.Similarly, no transcripts could be detected in the prog-eny of a cross between the strain possessing the amplifiedTLH1 gene and a second laboratory strain (Gao et al.2002). However, Yamada et al. (1998) showed that thehelicase gene on the yeast Y¢ element is expressed intelomerase-deficient yeast survivors, producing a pro-tein, Y¢-Help1, with helicase activity. However, the exactrole of this protein in the Y¢ amplification process is asyet unclear. The lack of a detectable TAH1 transcript inM. anisopliae could indicate that the TAS is now stablein the strain analysed and that the gene plays no role innormal cellular housekeeping. Moreover, the gene mayalso be subject to telomeric silencing (Gottschling et al.1990).

Phylogenetic analysis of selected RecQ-like helicasecores showed that the subtelomeric helicases of fila-mentous fungi form a separate clade, distinct fromother helicases. This topology probably reflects a sig-nificant difference between these two classes of heli-case, possibly related to the presumed mobile natureof the subtelomeric helicase genes. The position of thehelicase core relative to the whole protein appears tobe highly variable among the helicases included in ouranalysis. The helicases with core domains positionedtowards the N-terminal end tend to be smaller pro-teins, suggesting either redundancy of function of theN-terminal portions of those proteins that possessthem or that these regions are important for specificfunctions. It is known, for example, that the first 106amino acids of the 1,447-residue S. cerevisiae SGS1

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protein are essential for binding to DNA topoisom-erase III and that their coordinated interaction isimportant for their function in the processing of DNAafter exposure of cells to DNA-damaging agents(Bennett and Wang 2001; Hu et al. 2001). This dif-ference between M. anisopliae TAH1 and the othertelomere-associated helicase genes from U. maydis andM. grisea could be associated with the variation in thedistribution of the apparent insertions: the Metarhizi-um TAS is present at almost all telomeres and displayslittle RFLP. The shorter helicases encoded by theTASs of U. maydis and M. grisea could derive frominactive remnants of a similar element that have beenhomogenized by recombination.

The occurrence of ORFs for RecQ helicases in thesubtelomeric DNA of the filamentous fungi M. anisop-liae, U. maydis and M. grisea, and in the analogous Y¢element found in Saccharomyces yeasts, implies that thisclass of element could be an ancient addition to thefungal genome. However, while all laboratory strains ofS. cerevisiae and closely related species possess a Y¢element (Louis 1995; Naumov et al. 2000), strains of thesibling species S. bayanus–and yeast strains outside thisgroup of species–have no Y¢ (Naumov et al. 1992; Prydeand Louis 1997). Furthermore, the discontinuous dis-tribution of these subtelomeric elements among M. gri-sea and U. maydis isolates, and our data forMetarhizium, Gliocladium and Paecilomyces, suggestthat this character is not intrinsic to these fungi. Instead,it appears that it is the capacity for amplification ofTASs containing helicase genes that is evolutionarilyconserved in fungi.

Acknowledgements PWI was the recipient of a visiting post-doc-toral fellowship from the Brazilian National Research Council(Conselho Nacional de Desenvolvimento Cientifico e Tecnologico,CNPq) and MCVI was a post-doctoral CNPq visiting fellow atthe Department of Genetics, University of Leicester, UK duringthe period of this research. EJL is funded by The Wellcome Trust.The authors also thank EMBRAPA for additional financialsupport.

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