Two ITS forms co-inhabiting a single genet of an isolate of Terfeziaboudieri (Ascomycotina), a desert truffle

*Sharon Aviram, Nurit Roth-Bejerano and Varda Kagan-ZurThe Institutes for Applied Research, Ben-Gurion University of the Negev, POB 653, Beer-Sheva 84105,

*Israel; Author for correspondence (e-mail: zur@bgumail.bgu.ac.il)

Received 22 October 2002; accepted in revised form 21 November 2002

Key words: Heteroduplex, Heterokaryon, Intraspecific variability, Terfezia

Abstract

Two fruit-bodies of Terfezia boudieri Chatin, each exhibiting a mixture of two ITS-RFLP profiles, were found inthe Negev desert of Israel. A mycelial culture obtained from glebal out-growth maintained the double profile, asdid proliferating cultures established using single hyphae isolated from the original cultures. The main differencebetween the two ITS variants lies in a 21 bp deletion in the smaller variant. The question whether both variants arecontained within a single nucleus or occupy different nuclei sharing the same cytoplasm is discussed.

Introduction Pringle et al. 2000), or in mycelia of the binucleatefungal genus Ascochyta (Fatehi and Bridge 1998).

Below we describe the identification of two differ-As part of a project focussing on the truffles of the

ent ITS-RFLP patterns and the corresponding differ-Negev desert of Israel, we analysed the internal

ence in their sequences - in the nuclei of mycelialtranscribed spacer (ITS) of the nuclear rDNA gene

cultures derived from isolated single hyphae of thefamily of these fungi. Three RFLP (restriction frag-

desert truffle T. boudieri, Ascomycetes. Terfezias arement length polymorphism) patterns were discovered

presumed to bear uninucleate spores (Zhang 1992). Itin a population of Terfezia boudieri (as defined by J.

is commonly accepted that proliferating hyphae ofTrappe) (Holdengraeber et al. 2001).

uninucleate Ascomycetes do not normally form long-The ITS region is routinely used to study fungal

term heterokaryons (e.g. Glass and Kuldau (1992)).species (e.g. Gardes et al. (1991), Henrion et al.

However, analysis of the present results, backed by(1992, 1994), Paolocci et al. (1995), Buscot et al.

Volk and Leonard’s work on Morcchella (1990), and˚ ´(1996), Mello et al. (1996), Karen et al. (1997),

by our accompanying paper (Roth-Bejerano et al.Chillali et al. (1998), Paolocci et al. (1999)). It was

supports the existence of such long-termobserved some years ago that the ITS fragment does

heterokaryons.not always reveal a single RFLP pattern for eachspecies (e.g. Gardes et al. (1991), O’Donnell (1992)).Intraspecific ITS variability has since been described

Materials and methodsin samples from geographically separated populations(e.g. Gardes et al. (1991), O’Donnell (1992), Henrion

˚ ´et al. (1994), Karen et al. (1997)), within populations Biological materialcapable of sexual mixing in a single geographical

˚ ´region (Karen et al. 1997) and even among fungi of Fruit-bodies collected by Beduin during the 1997the same species infecting the root system of the same season from a field located in the western Negev,tree (Guillemaud et al. 1996). It has been shown that Israel, were defined to be Terfezia boudieri by J.different variants can occur in a single spore of Trappe. The fruit-bodies were used fresh, preservedarbuscular mycorrhizal fungi (Sanders et al. 1995; (in 30% ethanol, 30% glycerol and 40% deionized

20042004)

169 2004 Kluwer Academic Publishers. Printed in the Netherlands.Antonie van Leeuwenhoek 85: 169–174, 2004.

H O), or sun dried. Thin inner pieces of fresh Terfezia extracted from overnight cultures of the appropriate2

boudieri fruit-bodies containing unripe spores were bacterial strains using NucleoSpin Kit (Machery-surface-sterilised (dipped in 95% ethanol and fire Nagel, Duren, GmbH) and sent for sequencing.dried) and placed on Fontana agar (Bonfante-Fasolo For sequence analysis: The Big Dye Terminator kitand Fontana 1973) in Petri dishes. Within 4–6 w (Perkin-Elmer Inc., Branchburg, NJ) was used inhyphae started to emerge from some of the pieces; conjunction with the primer pair SP6 and T7 (Prom-these were collected and transferred to fresh media. ega, Madison, WI, USA ) to sequence the ITS frag-Single hyphae were isolated from such cultures by ment directly from each of two plasmids, containing amarking the location of individual hyphae under the different 42a ITS form. Primers were synthesized atmicroscope, then cutting out the appropriate agar the Molecular Biology Lab. Hadassa Hospital,pieces aseptically and transferring them singly to Jerusalem. The sequence was determined using anfresh medium. The excised pieces were then verified ABI prism 377 DNA sequencer (Applied Biosystemunder the microscope. Inc., Foster City, CA) at the sequencing unit of the

Hebrew University of Jerusalem, Israel.DNA extraction and PCR amplification

GenBank accession no.The method described by Grube et al. (1995), whichinvolves the use of guanidine extraction buffer, Terfezia boudierii clone 42a1 (small) - AF301418.phenol extraction, and NaI 1 glassmilk DNA precipi- Terfezia boudierii clone 42a2 (large) - AF301419.tation, was employed to obtain small amounts ofamplifiable DNA. About 100 mg of a fresh fungal Restriction reactionssample was usually sufficient. DNA was amplifiedusing primers ITS 1 and ITS 4 (White et al. 1990). In a total volume of either 15 or 25 ml, either 10 or 20The primers were synthesized by Dr. M. Huleihel at ml of the PCR products or plasmid preparations werethe Institutes for Applied Research, BGU, Israel. ITS combined with 1/10th volume of the buffer providedamplification was performed as described by Kagan- by the manufacturer and 1–3 units ITS of Hinf I (MBI,Zur et al. (1999). Fermentas, Lithuania). Reactions were performed at

37 8C for 4–6 h and stopped by adding 1/10 volumeCloning and sequence analysis of gel loading buffer. Mixtures: ITS amplification

products of single-profiled specimens were mixed andPCR amplified ITS fragments of the 42a double either denatured (at 95 8C for 10 min) and allowed toprofiled culture were cloned into pGEM - T easy kit re-anneal before further handling, or not denatured.(Promega, Madison, WI, USA) in accordance with the Restriction reactions were performed after mixing.manufacturer’s recommendations. The plasmid mix- Restriction products were separated on 3.0% regularture was transformed into E. coli XL1-blue (Bullock agarose (Pronarose D1, Hispanagar, Spain). Gelset al. 1987) competent cells by electroporation at 2.5 were post stained with ethidium bromide and photo-KVolts, 200 Ohm for a few seconds and, following 1 h graphed under UV light (Compact B.I.S. camera,of incubation in Eppendorf tubes at 37 C, spread over Renium, Jerusalem) and printed (Thermal ImagerLB (Miller 1972) plates containing 100 mg/ml am- FTI-500, Renium, Jerusalem).picillin. Single colonies were picked up and grown inliquid LB overnight. Plasmids were extracted fromthese cultures using the alkaline method (Birnboim Resultsand Doly 1979). Plasmid preparations were cut byEcoRI (MBI, Fermentas, Lithuania) as described Out of the 130 fruit-bodies analysed, two-42a andbelow. The resulting products were separated on 59-exhibited a complex ITS profile upon digestion byagarose gels (see below) to yield a linear plasmid Hinf I (Figure 1). There were more fragments thanfragment and an insert. Plasmids containing inserts expected, and the sum of their sizes was greater thanwere subjected to direct ITS amplification using ITS that of the ITS fragment actually observed, as if theprimers. The fragments were then digested using single lobe contained a mixture of at least two ITSHinf I restriction enzymes and the resulting RFLP was forms (Figure 1). At first we assumed that the fruit-analysed. Plasmids containing relevant inserts were bodies under study harbored two genotypes of hy-

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phae. However, after two years of sub-culturing fromdifferent sites on a Petri dish containing a mycelialculture of truffle 42a, we observed that the culture hadmaintained the same complex profile. As the putativeITS types did not segregate, we surmised that theymight be contained within single hyphae. To test this,we proceeded to cut away pieces of agar with singlehyphae from the edges of growing cultures and toculture them individually (Figure 2). Out of 20 agarpieces, 10 turned out to contain more than one hypha,and several hyphae failed to develop or grew only to alimited extent. However, two cultures of single hy-phae continued to proliferate and to yield independentcultures. Analysis revealed that each mycelium de-veloped from a single hypha had maintained theobserved complex profile.

Comparison against profiles we had recorded ear-lier (Holdengraeber et al. 2001) confirmed that threeof the five fragments observed in the complex profile

Figure 2. A single isolated hypha. A: immediately after isolation.fragments belonged to form 2, but could not fully B: one day later. Scale bar (8 mm) 5 1 mmaccount for the other two. We therefore judged furtheranalysis to be necessary.

To this end, ITS fragments of 42a were cloned into previously described form 2 (Holdengraeber et al.pGEM and introduced into E. coli strain XL1-blue. 2001). It may thus be concluded that 42a1 constitutesTwo sizes of inserts were recovered and one clone of a fourth form.each type was sequenced. The smaller form, termed For a final proof that the cloned fragments were42a1 - AF301418, and the larger form, termed 42a2 - what we were looking for - ITS fragments of pureAF301419 (Figure 4), differed only by a deletion of 42a1 and 42a2 were mixed before performing Hinf I21 bp. The 42a2 fragment closely resembled the

Figure 1. Hinf I digested ITS-RFLP profiles of several Terfezia Figure 3. Comparison of ITS-RFLP profiles of forms representing aboudieri fruit bodies. Fb5: Fruit-body No. 5, a Form 2 profile. mixture of 42a1 and 42a2 against the 42a profile. Lane 1: FormFb44: Fruit-body No. 44, a Form 2 profile. Fb59: Fruit-body No. 42a1. Lane 2: Form 1. Lane 3: Form 2 (42a2). Lane 4: Original, not59, an unexpected profile. Fb39: Fruit-body No. 39, a form 1 cloned, 42a. Lanes 5: Mixed and denatured 42a1 1 42a2. Lane 6: Aprofile. Lane Fb42a: Fruit-body No. 42a, an unexpected profile. non-denatured mixture of 42a1 1 42a2.

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Figure 4. Sequence of the 42a2 ITS fragment. Non-numbered sequences denote primer sequences that were not part of the actual sequenceobtained. Underlined sequence is the sequence deleted in 42a1

digestion. As expected from such mixtures, only 4 one of the following mechanisms: a. by pseudo-bands were detected when the procedure was carried homothallism (e.g. Nelson (1996), Coppin et al.out without denaturing (Figure 3, lane 6). However, (1997)); b. through anastomoses between vegetativewhen the mixture was denatured, cooled, cut, and hyphae sharing the same mating types and the sameseparated on gel, an extra band, a heteroduplex het and vic allels to form a compatible heterokaryon(Selosse et al. 1996), absent in profiles of the non- (Wu et al. 1998); c. via tol mutations (Coppin et al.denatured mixture, made an appearance (Figure 3, 1997; Shiu and Glass 1999). No other mechanismlane 5); the result was a precise reconstruction of the leading to viable vegetative heterokaryons of as-42a profile (Figure 3, lane 4). comycetes is known, while sexual heterokaryons are

short-lived and are limited to hypha(e) developingfrom the ascogonium after fertilization (e.g. Glass and

Discussion Kuldau (1992)). Nothing is known concerning stableheterokaryons in Tuberaceae. However, one study at

The internal transcribed spacer region (ITS) of the least supports the existence of a stable Tuber melanos-rRNA gene cluster is considered to be sufficiently porum dikaryotic state (Bonfante-Fasolo and Brunelvariable to enable discrimination between species, 1972). In an accompanying paper (Roth-Bejerano etand, in most cases, this expectation has been justified

˚ ´(e.g. Buscot et al. (1996), Chillali et al. (1998), Karen between monosporic and glebal hyphae of a Terfeziaet al. (1997)). However, intraspecific variability species, which suggests a normal long term existenceseems to be quite common in fungi, and fungal of an ascomycetous heterokaryon.variants have been reported within mixing fungal In light of our finding that the two 42a forms arepopulations of a species (e.g. Sanders et al. (1995), identical except for a deletion, we cannot rule out theGuillemaud et al. (1996), Fatehi and Bridge (1998), possibility that both forms are occupying the samePringle et al. (2000)). haploid nucleus as the result of an in-nucleus deletion

Two coexisting forms could be maintained in two affecting some of the rRNA gene repeats, but notseparate nuclei to produce a stable heterokaryon by others. The coexistence of two ITS forms within one

al. 2004) we show a difference in nuclei pairing

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1998. Delineation of the European Armillaria species based onnucleus has not been demonstrated so far. In our case,the sequences of the internal transcribed spacer (ITS) of ribosom-the two forms may be expected to be subject to theal DNA. New Phytol. 138: 553–561.processes of molecular drive (Dover 1982, 1986),

Coppin E., Debuchy R., Arnaise S. and Picard M. 1997. Matingleading the one to overtake the other in time. Since the types and sexual development in filamentous Ascomycetes.differences between the variants are too slight to Microbiol. Mol. Biol. Rev. 61: 411–428.

Dover G. 1982. Molecular drive: a cohesive mode of speciesenable the design of specific primers or markers at thisevolution. Nature 299: 111–117.stage, we lack a molecular tool with which to evaluate

Dover G. 1986. Molecular drive in multigene families: how bio-these two possibilities. However, within the five yearslogical novelties arise, spread and are assimilated. Trends Genet.

in which this mycelium is maintained and subcultured 2: 159–165.every two weeks, no change in the intensity of any of Fatehi J. and Bridge P. 1998. Detection of multiple rRNA-ITSthe bands was observed. In view of this observation, regions in isolates of Ascochyta. Mycol. Res. 102: 762–766.

Gardes M., White T.J., Fortin J.A., Bruns T.D. and Taylor J.W.and of the sound possibility of a long-term dikaryotic1991. Identification of indigenous and introduced symbioticexistence of a Terfezia mycelial culture, we surmisefungi in ectomycorrhizae by amplification of nuclear and mito-

that the two ITS forms are maintained in separate chondrial ribosomal DNA. Can. J. Bot. 69: 180–190.nuclei. Glass N.L. and Kuldau G.A. 1992. Mating type and vegetative

In conclusion, we have shown that the unusual incompatibility in filamentous ascomycetes. Ann. Rev.Phytopatol. 30: 201–224.Hinf I digest-profile of Terfezia boudieri truffle 42a

Grube M., Depriest P.T., Gargas A. and Hafellner I. 1995. DNAresults from the coexistence of two ITS forms sharingisolation from lichen ascomata. Mycol. Res. 99: 1321–1324.

the same cytoplasm, and also that the two coexisting Guillemaud T., Raymond M., Callot G., Cleyet-Marel J.-C. andforms are identical apart from a 21 bp deletion. We Fernandez D. 1996. Variability of nuclear and mitochondrialhave also shown that the extra band visible in this ribosomal DNA of a truffle species (Tuber aestivum) 100: 545–

550.strain’s profile is formed upon denaturation and re-Henrion B., Chevalier G. and Martin F. 1994. Typing truffle speciesannealing (taking place during the PCR process) and

by PCR amplification of the ribosomal DNA spacers. Mycol.is therefore a heteroduplex (Selosse et al. 1996). We Res. 98: 37–43.argue that the two forms are maintained in separate Henrion B., Le-Tacon F. and Martin F. 1992. Rapid identificationnuclei, in a long-term heterokaryon. of genetic variation of ectomycorrhizal fungi by amplification of

ribosomal RNA genes. New Phytol. 122: 289–298.Holdengraeber S., Martin F., Roth-Bejerano N. and Kagan-Zur V.

2001. Some lobed fruit bodies of Terfezia boudieri (a desertAcknowledgements truffle) combine two ITS; RFLP patterns, suggesting independent

´ ´origins In: Federation Francaise des Trufficulteurs (FFT) eds.Proceedings of the Vth International Congress, Science andWe wish to thank Dr. Marc-Andre Selosse for useful

comments.78–81.

Kagan-Zur V., Kuang J., Tabak S., Taylor F.W. and Roth-BejeranoN. 1999. Potential verification of a host plant for the desert truffle

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