Hexose uptake in the plant symbiotic ascomycete Tuber borchii Vittadini: biochemical features and...

Fungal Genetics and Biology 44 (2007) 187–198

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Hexose uptake in the plant symbiotic ascomycete Tuber borchii Vittadini: biochemical features and expression

pattern of the transporter TBHXT1

Emanuela Polidori a, Paola Ceccaroli b, Roberta Saltarelli b, Michele Guescini b, Michele Menotta b, Deborah Agostini b, Francesco Palma b, Vilberto Stocchi a,b,¤

a Istituto di Ricerca sull’Attività Motoria, Università degli Studi di Urbino “Carlo Bo”, Via I Maggetti 26, 61029 Urbino (PU), Italyb Istituto di Chimica Biologica “Giorgio Fornaini”, Università degli Studi di Urbino “Carlo Bo”, Via A. SaY 2, 61029 Urbino (PU), Italy

Received 4 May 2006; accepted 1 August 2006Available online 26 September 2006

Abstract

Here, we report the Wrst evidence of a hexose transporter gene, Tbhxt1, in the ectomycorrhizal ascomycete Tuber borchii Vittadini. Theprotein encoded by Tbhxt1 functionally complements the hxt-null mutant Saccharomyces cerevisiae EBYVW.4000. TBHXT1 has a strongpreference for D-glucose (KmD 38§ 10 �M) over D-fructose (KmD 16§ 5 mM) and uncoupling experiments indicate that TBHXT1 cata-lyzes the transport via a proton-symport mechanism. The investigations on the substrate speciWcity reveal that TBHXT1 also importsD-mannose, and the use of deoxyglucose analogues shows that the hydroxyl groups at C1, C3 and C4 are important for substrate recogni-tion. Tbhxt1 is not regulated by fructose, but it reaches its highest level of expression at 3 mM glucose and is repressed by very highglucose concentration. Prolonged carbon starvation condition upregulates Tbhxt1, while its expression remains at basal level in theectomycorrhizal tissue. The mode of regulation of Tbhxt1 is consistent with its role as a high-aYnity D-glucose transporter.© 2006 Elsevier Inc. All rights reserved.

Keywords: Tuber borchii; Ectomycorrhizal fungus; Hexose transporter

1. Introduction

The Wrst step in carbohydrate metabolism is the uptakeof the appropriate molecules by cells. For this purpose,microorganisms use a variety of diVerent membrane-boundtransport proteins of the Major Facilitator Superfamily(MSF), a group which appears to share a common ances-tral origin (Andre, 1995). Hexose transporters have beeninvestigated for a variety of organisms including bacteria,fungi, plants and humans.

The eukaryotic fungal system in which sugar transporthas been the most comprehensively studied is the yeast Sac-charomyces cerevisiae (Boles and Hollenberg, 1997). In this

* Corresponding author. Fax: +39 0722 305262.E-mail address: [email protected] (V. Stocchi).

1087-1845/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.fgb.2006.08.001

organism, 18 genes, HXT1–HXT17 and GAL2, encode pro-teins belonging to the monosaccharide transporter family,although the hexose transporters encoded by HXT1–7 andGAL2 are the major transporters of yeast. Two other pro-teins, called Snf3 and Rgt2, act as glucose sensors and con-trol the expression of the hexose transporters according tothe availability and concentration of carbon sources. Thus,there are eVectively two glucose uptake systems in yeast, aconstitutive low-aYnity system and a glucose-repressed high-aYnity system (Lagunas, 1993; Özcan and Johnston, 1999).

Sugar transporters have been also studied in Wlamentousfungi. In the saprophytic fungus Neurospora crassa, there aretwo glucose uptake systems: a constitutive low-aYnity trans-port system and a glucose-repressible high-aYnity transportsystem (Scarborough, 1970; Schneider and Wiley, 1971). Sev-eral putative hexose transporters also have been identiWed inthe genome sequence (Galagan, 2003) and one of them, hgt-1,

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188 E. Polidori et al. / Fungal Genetics and Biology 44 (2007) 187–198

has been identiWed and characterized as a high-aYnity glu-cose transporter (Xie et al., 2004). In Aspergillus nidulans,both high- and low-aYnity glucose transport systems havebeen identiWed using transport assays (Mark and Romano,1971). The A. nidulans genome contains at least 17 putativehexose transporters, one of which, hxtA, is a high-aYnityhexose transporter involved in sugar metabolism duringsexual development (Wei et al., 2004). The mstA gene fromAspergillus niger encodes a high-aYnity sugar/H+ symporterand is subjected to carbon catabolite repression and pH regu-lation. Disruption of mstA did not result in a discernible phe-notype compared with the isogenic reference strains,indicating that A. niger possesses other sugar transporterswhich can catalyze the uptake of diVerent monosaccharides(vanKuyk et al., 2004). In the mycoparasitic fungus Tricho-derma harzianum, gtt1 encodes a high-aYnity glucose trans-porter (Delgado-Jarana et al., 2003). This gene is repressedby high levels of glucose and induced under low carbon con-centrations and starvation conditions.

Little is known about sugar transport in biotrophicplant-interacting fungi. To date, only two genes encodingmonosaccharide transporters have been cloned and func-tionally characterized; both are from basidiomycetes: hxt1from the rust fungus Uromyces fabae and AmMst1 fromthe ectomycorrhizal fungus Amanita muscaria. hxt1 isexpressed in haustoria, and there was no evidence for otherhexose transporters in any of the other developmentalstages examined (Voegele et al., 2001). AmMst1 from A.muscaria is constitutively expressed in fungal hyphae underall growth conditions and shows an increased expression athigh levels of monosaccharides (Nehls et al., 1998). Thegenes are closely related and have a higher preference forglucose than for fructose uptake. Recently, another A. mus-caria hexose transporter has been described (Nehls, 2004)and a number of highly similar hexose transporter genesfrom the basidiomycete Paxillus involutus have been identi-Wed using cDNA microarrays (Le Quere et al., 2006). Nev-ertheless, at the moment these genes have not beeninvestigated at the functional level.

At present, there is no available information on hexoseuptake by ascomycetous ectomychorrhizal fungi, like Tuberborchii Vittadini. This fungus forms ectomycorrhizae with avarious host plant (Zambonelli and Branzanti, 1989) and inthe plant–fungus symbiotic interface, fungal and root corti-cal cells compete for monosaccharides, glucose and fruc-tose, generated from the hydrolysis of plant-derived sucrose(Nehls et al., 2000; Wright et al., 2000).

As such, we aimed to study sugar assimilation in T. bor-chii by cloning and functional characterizing Tbhxt1, whichcodes for the Wrst hexose transporter in a plant symbioticascomycete. Interestingly, Tbhxt1 regulation diVers fromother sugar transporters belonging to plant—interactingfungi, since it responds to variation in the glucose concen-tration but is not regulated by the symbiotic stage. Theavailability of cloned and functionally characterized sugartransporters from ectomycorrhizal fungi represents anessential prerequisite in order to gain a molecular under-

standing of the functioning and regulation of sugar uptakein these ecologically and agronomically important microor-ganisms.

2. Materials and methods

2.1. Biological materials and growth conditions

Tuber borchii mycelia (MYA-1019) (Zambonelli et al.,1995) were Wrst cultured on Melin–Norkrans liquidmedium (MMN) (pH 6.6) (Molina, 1979) containing10 mM glucose as the sole carbon source (20 days) and thentransferred (3 days) to liquid MMN medium containingglucose or fructose at concentrations ranging from 0.1 to50 mM or no carbon source (3, 7 and 20 days of starvation).Mycelia were cultured in 100 ml Xasks, each containing70 ml of medium inoculated with plugs of mycelia fromPotato Dextrose Agar (PDA) plates, and kept in a growthchamber at 24 °C in the dark with no agitation.

Tuber borchii–Tilia platyphyllos ectomycorrhizae wereobtained as previously described (Polidori et al., 2002).

In this study, we used the S. cerevisiae strainEBYVW.4000 (MATa �hxt1-17 �gal2 �stl1 �agt1 �mph2�mph3 leu2-3, 112 ura3-52 trp1-289 his3-�1 MAL2-8c

SUC2) (Wieczorke et al., 1999) and the yeast expressionvector p426HXT7-6His provided by Prof. E. Boles (J.W.Goethe University, Frankfurt). The yeast mutant strain wasmaintained and grown as described by Boles (Boles andHollenberg, 1997).

2.2. Hexose uptake experiments in Tuber borchii mycelium

Tuber borchii mycelia were grown in the dark at 24 °C onmodiWed MMN solid medium containing 0.1, 3 or 50 mMof hexose (glucose or fructose). To enable labeled hexoseuptake experiments, mycelial cultures were grown on semipermeable cellophane membranes (Bio-Rad, USA). In theassay, fungal discs were cut from actively growing 20-days-old colonies and incubated at 24 °C for 15 min in 1 ml of aliquid MMN medium sugar-free supplemented with 3 �MD-[U-14C]glucose (speciWc activity 11.7 GBq/mmol) or 3 �MD-[U-14C]fructose (speciWc activity 8.69 GBq/mmol).

The fungal discs were washed twice in a Millipore Wlterbox with 10 ml of distilled water and then counted in aPackard liquid scintillation counter Tri-Carb 2100TR(Perkin-Elmer).

2.3. Isolation of nucleic acids

Genomic DNA was isolated from T. borchii mycelia fol-lowing the protocol of Erland et al. (1993). Total RNA wasisolated from mycelia and T. borchii–T. platyphyllos ecto-mycorrhizae by using an RNeasy Plant mini kit (Qiagen),following the manufacturer’s instructions. The Wnal con-centration and the quality of the nucleic acids were evalu-ated both spectrophotometrically and by agarose gelelectrophoresis.

E. Polidori et al. / Fungal Genetics and Biology 44 (2007) 187–198 189

2.4. Isolation and molecular characterization of the Tbhxt1 cDNA

A pair of degenerate oligonucleotides Nfor (5�-YTICARCARYTIACIGG-3�) and Nrev (5�-ARISWIAWIARICCYTTIGTYTC-3�), previously described by Nehlset al. (1998), and a new primer pair based on the multiplealignment of known fungal monosaccharide transporters(TraspF: 5�-CCNGARTCNCCNMGNT-3� and TraspR:5�-RAARTTNAYNARCCARTT-3�) were used in PCRexperiments in all possible pairwise combinations with T.borchii genomic DNA and cDNA. PCRs were made in aWnal volume of 50�l containing 1£ reaction buVer, 2 mMMgCl2, 100 �M dNTPs, 100 pmol of each primer selectedand 0.5 U of Taq DNA polymerase (Qiagen). The mixturewas incubated for 5 min at 94 °C and then subjected to 35cycles of 30 s at 94 °C, 45 s at 38 °C and 1 min at 72 °C, witha Wnal step of 7 min at 72 °C. A fragment of the expectedsize (V500 bp) was cloned into pGEM-T vector (Promega),sequence-veriWed and used as a homologous probe forscreening a cDNA library from 30-day-old T. borchii myce-lium (Sambrook and Russell, 2001). A cDNA of 1842 bp,called Tbhxt1 (GenBank Accession No. AY956320), wasisolated, sequenced on both strands and compared againstthe data bank protein sequences with the BLASTX pro-gram (Altschul et al., 1990).

Phylogenetic analysis was performed with hexose trans-porter sequences obtained from GenBank databases. Mul-tiple alignment was carried out using ClastalW (Version1.83, http://www.ebi.ac.uk/clustalw/) with the followingparameters: matrix, pam; gap open, 5; gap extension, 0.5.The phylogenetic tree was constructed with the maximumparsimony algorithm implemented on the Protpars pro-gram of the Phylip package version 3.65 (Felsenstein, 2005).Support for the inferred clades was obtained by bootstrapanalysis from 100 replications of the data set using the Seq-boot and Consense programs. Phylogenetic tree was dis-played and edited with TreeView (Page, 1996). Theaccession numbers of sequences used in the alignment aregiven in Fig. 3. Sequence analyses and homology searcheswere performed using the online blast suite of programs(NCBI, USA).

2.5. Southern blotting

Ten micrograms of T. borchii mycelium genomic DNAwere digested with the enzymes EcoRI, HindIII and PstI,which do not cut the Tbhxt1 gene used as a probe. Thedigested DNA was electrophoresed on a 0.8% agarose gel,blotted onto version 2.0 Hybond™-N+ positively chargednylon membranes (Amersham Life Science) in accordancewith the manufacturer’s instructions and hybridised withTbhxt1, which was 32P-labeled using the RediPrime Label-ing Kit (Amersham Life Science). The Wlter was washedunder low (2£ SSC and 0.1% SDS at 60 °C) and high(0.1£ SSC and 0.1% SDS at 60 °C) stringency washingconditions.

2.6. Heterologous complementation in the yeast mutant strain EBYVW.4000

For heterologous expression in S. cerevisiae, the codingregion of Tbhxt1 was ampliWed from the excised pBlue-Script-Tbhxt1 plasmid using two primers containing therestriction sites for SpeI and ClaI, as forward and reverseprimers, respectively (Tbhxt1SpeI-f: 5�-GACTAGTGGTTTCATCATCAAGAGACCAGAGG-3� and Tbhxt1ClaI-r:5�-CCATCGATGGCTAAACCTCGCCGTGCCC-3�), andthe high Wdelity Platinum Taq DNA Polymerase (Invitro-gen). The ampliWcation product was subcloned intopGEM-T vector (Promega), sequence-veriWed and digestedwith the enzymes SpeI and ClaI. Then it was ligated usingT4 DNA Ligase (Promega) into the yeast expression vectorp426HXT7-6His, previously treated with the sameenzymes. Escherichia coli XL1-Blue cells were transformedwith the construct and the recombinant clones were identi-Wed by PCR using Tbhxt1 speciWc primers. Plasmid DNAof p426HXT7-Tbhxt1 was isolated using a plasmid extrac-tion kit (Qiagen) and the correct insertion of the cDNA wastested by sequencing the cDNA/vector junctions. The con-struct p426HXT7-Tbhxt1 was transformed into chemicallycompetent cells of S. cerevisiae strain EBYVW.4000. Yeasttransformants were Wrst selected for their ability to grow onthe selective YNB medium containing the appropriateamino acids (Boles and Hollenberg, 1997) and 1% maltose.Some of them were subsequently transferred to the samemedium containing glucose or fructose at a concentrationof 2% or 0.2% as the sole carbon source. The growth of theyeast strain expressing Tbhxt1 was compared with that of astrain harbouring the plasmid p426 without insert. Oneclone, able to grow on glucose, was chosen for the addi-tional experiments.

2.7. Functional assays in Saccharomyces cerevisiae

Recombinant yeasts were grown in YNB 1% maltoseselective medium (Boles and Hollenberg, 1997) to anOD600D 0.5–0.7, harvested by centrifugation, washed twicewith 100 mM potassium-phosphate buVer (pH 6.5) andresuspended in the same buVer to a Wnal concentration of109 cell/ml. Sugar uptake started mixing 100 �l of yeast cellsuspension with 100 �l of glucose (25�M–10 mM nonradioactive glucose plus 3 �M D-[U-14C]glucose) or fructose(0.25–50 mM non radioactive fructose plus 3�M D-[U-14C]fructose). Ten microliters of each reaction mixture wasused for the evaluation of the total radioactivity while theremaining samples were incubated at 30 °C for 1 min. Oncesugar uptake was blocked by the addition of 10 ml of ice-cold 100 mM potassium-phosphate buVer (pH 6.5) contain-ing 500 mM unlabeled glucose or fructose, the cells wereWltered onto a Millipore Wlter boxing through Hybond-Cextra Wlters (Amersham), and washed twice with 10 ml ofice-cold distilled water. The Wlters were then transferredinto scintillation vials containing 5 ml of scintillant andradioactivity was measured with a Packard liquid

http://www.ebi.ac.uk/clustalw/

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scintillation counter. The amount of glucose or fructoseretained on the Wlters was calculated from the speciWc activ-ity of the substrate solutions used during the experiments.

All uptake experiments were repeated with independentsamples at least three times. The kinetic parameters weredetermined using the Lineweaver–Burke plot, and all thedata were analysed by non linear regression using the Enz-Fitter program. The standard errors were evaluated by thesame program.

For the hexose consumption measurements, the cellstreated as previously described were resuspended in100 mM potassium-phosphate buVer (pH 6.5) containingglucose or fructose at a Wnal concentration of 1 mM. Thecells were incubated at 30 °C and removed at 0, 15, 30, 60,90 and 120 min and immediately centrifuged at 8000g. Thehexose concentration of the supernatant was measuredspectrophotometrically at 37 °C in a system coupled withhexokinase, glucose-6-phosphate dehydrogenase and phos-phogluco isomerase as described by Beutler (1984).

For the substrate speciWcity of TBHXT1, the uptake ofglucose was investigated in the presence of diVerent unla-beled substrates: deoxyglucose analogues (DOG) [1DOG,2DOG, 3DOG, 5-thio-D-glucose (5TG), 6DOG], D-glucose,D-fructose, D-mannose, 3-O-methylglucose (3OMG), D-galactose, xylose, arabinose, sorbitol and mannitol. Theconcentration of the unlabeled competing substrates was100 times higher than glucose Km.

The sugar uptake inhibition was tested using the uncou-pling compounds carbonyl cyanide m-chlorophenylhydraz-one (CCCP) and 2,4-dinitrophenol (DNP) dissolved inethanol and sodium orthovanadate at a Wnal concentrationof 100 �M, respectively, in the presence of 38�M D-[U-14C]glucose.

2.8. Quantitative real-time PCR (qRT-PCR)

One microgram of DNase (Ambion)-treated total RNAwas reverse transcribed as described by Guescini et al.(2003). RNAs extracted from T. borchii mycelia, grown aspreviously described, and from T. borchii–T. platyphyllosectomycorrhizae were used as samples for reverse transcrip-tion. T. borchii 18S rRNA (tb18S) was employed as aninternal standard. SpeciWc primers for Tbhxt1 (TbhxtF:5�-ATTGCCTCTTGCCTGGTTTTCT-3� and TbhxtR:5�-CAATTACACGACCAGCAGCCATA-3�) and tb18S (TB18SF:5�-ACTGGTCCGGTCGGATCTT-3� and TB18SR:5�-TTCAAAGTAAAAGTCCTGGTTCCC-3�) were designedto amplify under the same cycling conditions and proce-dure reported in Guescini et al. (2003), generating productswith sizes ranging from 86 to 167 bp. PCR was performedin a Bio-Rad iCycler iQ Multi-Color Real-Time PCRDetection System. The speciWcity of the ampliWcation prod-ucts obtained was conWrmed by examining thermal dena-turation plots and by sample separation in a 3% DNAagarose gel. The amount of the target transcript was relatedto that of the reference gene using the method described byPfaXl (2001). Each sample was tested in triplicate by quanti-

tative PCR, and the samples obtained from at least six inde-pendent experiments were used to calculate the means andstandard error.

3. Results

3.1. Hexose uptake in Tuber borchii mycelium

The hexose uptake of T. borchii hyphae was initiallytested by in vivo experiments in mycelia grown at very low(0.1 mM), low (3 mM) and high (50 mM) glucose or fructoseconcentrations. As shown in Fig. 1, T. borchii myceliumexhibits a preference for glucose over fructose. Further-more, the glucose and fructose uptake was signiWcantlyincreased when the mycelium was grown at low hexose con-centrations.

3.2. Cloning and molecular characterisation of Tbhxt1

As a Wrst step towards the cloning of T. borchii hexosetransporter gene, we cloned a 500 bp DNA to use as homol-ogous probe for screening a T. borchii mycelium cDNAlibrary. The sequence analysis of this fragment was carriedout using the BLASTX program available on the NCBIwebsite (http://www.ncbi.nlm.nih.gov) and showed a highsimilarity with many fungal species’ monosaccharide trans-porter genes. The screening of the T. borchii myceliumcDNA library with the cloned fragment resulted in Wvehybridising phage clones. They were all converted intopBluescript SK¡ plasmids and sequence-veriWed. Two ofthem were false positives, whereas the other three resulted

Fig. 1. Uptake of glucose (gray columns) and fructose (black columns) inTuber borchii mycelium. The mycelia were grown either at 0.1, 3 or 50 mMglucose or fructose, and transferred into sugar free medium. Then 3 �Mlabeled glucose or fructose were added and uptakes were measured after15 min of incubation. G0.1, glucose 0.1 mM; G3, glucose 3 mM; G50, glu-cose 50 mM; F0.1, fructose 0.1 mM; F3, fructose 3 mM; F50, fructose50 mM.

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identical in length (1842 bp) and corresponded to a gene,Tbhxt1, with a high degree of similarity to glucose/hexosetransporter-encoding gene. The sequence was submitted tothe GenBank database under the Accession No. AY956320.Tbhxt1 contains an open reading frame of 1563 bp codingfor a putative protein of 520 amino acids with a predictedmolecular mass of 59.85 kDa. A comparison of the genomicand cDNA sequences obtained with three diVerent pairs ofprimers selected on the basis of the complete Tbhxt1cDNA, highlighted the presence of three introns with typi-cal eukaryotic gene intron consensus sequences (Breathn-ach et al., 1978). They are all localised on the 5� side andhave a length of 114, 127 and 64 nt, respectively (Fig. 2A).Southern analysis revealed only one hybridisation signalboth in low- and high-stringency washing conditions, sug-gesting that Tbhxt1 is a single-copy gene in T. borchiigenome (Fig. 2B).

The highest similarities of the deduced protein werefound with several fungal hypothetical proteins (N. crassaXP_959411, Aspergillus oryzae BAE55190, A. nidulansCAJ44796, Gibberellae zeae XP_382817 and Magnaporthegrisea XP_369261) which are supposed to be putative hex-ose transporters. Among the functionally characterizedhexose transporters the TBHXT1 polypeptide shares max-imum identity (52%) with the MST1 transporter of theectomycorrhizal fungus A. muscaria (CAB06078) (Nehlset al., 1998), HXT1p of the basidiomycete U. fabae(CAC41332) (Voegele et al., 2001), followed by RCO-3 ofN. crassa (AAA99806, 50% of identity) (Madi et al., 1997).The maximum parsimony tree consisting of 44 full-length

polypeptide sequences of hexose transporters from diVer-ent fungal species shows that all TBHXT1-relatedsequences cited above cluster in the same branch with abootstrap value of 100% and all the hypothetical proteinsfall on this branch with a bootstrap value of 67% (Fig. 3).Phylogenetic analysis using the neighbour-joining methodalso produced a similar tree of the same topology (data notshown).

The putative TBHXT1 membrane topology deducedusing several programs, TMHMM2.0 (Sonnhammer et al.,1998), SOSUI (Hirokawa et al., 1998), TMPRED (Hof-mann and StoVel, 1993) and HMMTOP (Tusnády andSimon, 2001), was predicted to have 12 membrane-span-ning domains which form the polypeptide backbone, with alarger cytoplasmic loop between the sixth and the seventhtransmembrane helix and N and C termini located on thecytoplasmic face. Two G-R-[KR] motifs (Maiden et al.,1987), typical of these transport proteins, were identiWed.The Wrst one is located between the second and third trans-membrane domains, at the amino acid positions 91–93, andthe second between transmembrane domains 8 and 9, at theamino acids 333–335. Moreover, two other patterns werefound along the protein sequence: the Wrst in the loopbetween transmembrane domains 4 and 5, [LIVMF]-x-G-[LIVMFA]-x(2)-G-x(8)-[LIFY]-x(2)-[EQ]-x(6)-[RK] at theamino acid positions 125–150 (IaGLgvGlvsalvplYqsEsap-kwiR), and the other [LIVMSTAG]-[LIVMFSAG]-x(2)-[LIVMSA]-[DE]-x-[LIVMFYWA]-G-R-[RK]-x(4,6)-[GSTA], between amino acids 325 and 342 (GLwmVEkMGRRnlllfgA) (Fig. 4).

Fig. 2. In (A) schematic representation of Tbhxt1 deduced exon–intron structure (dotted lines: 5� and 3� UTRs; thick lines: introns; black blocks: exons)and schematic representation of the corresponding putative protein (white blocks: transmembrane helices). In (B) Southern blotting analysis. Final wash-ing conditions were 0.1£ SSC and 0.1% SDS at 60 °C.

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Fig. 3. Phylogenetic relationships of TBHXT1 to diVerent hexose fungal transporters. The maximum parsimony tree is based on a multiple alignmentof TBHXT1 to 43 proteins of diVerent fungal species retrieved from database. S. cerevisiae AAA34700 was used as outgroup taxon to root the tree.The bootstrap support (SEQBOOT program, phylip package) for each branch (100 replications) is shown. The symbol � indicates putative hexosetransporters.


3.3. Functional properties of TBXHT1

In order to investigate the kinetic properties and sub-strate speciWcity of TBHXT1 the gene was cloned intothe yeast expression vector p426HXT7-6His, under thecontrol of the HXT7 promoter (Boles and Hollenberg,1997). The resulting plasmid (p426HXT7-Tbhxt1) andthe vector alone were transformed into S. cerevisiaestrain EBYVW.4000 lacking all 20 hexose transporters.Growth of these strains was tested after 4 days at 30 °Con plates containing 2% (w/v) glucose or fructose as thesole carbon source, and only yeast clones expressingTbhxt1 cDNA were able to restore the growth ofEBYVW.4000 on solid medium containing one of thesugars mentioned above (Fig. 5A). Growth experimentsin liquid medium yielded similar results, and no diVer-ence in growth was observed on 0.2% glucose comparedwith 2% glucose or on 0.2% fructose compared with 2 %fructose (data not shown).

Extracellular hexose consumption experiments con-Wrmed that the expression of TBHXT1 in S. cerevisiaestrain EBYVW.4000 enabled it to consume glucose or fruc-tose, whereas the reference strain, containing the expressionvector alone, did not show any uptake of these two hexosesugars (Fig. 5B).

The uptake of both glucose and fructose intop426HXT7-Tbhxt1 transformed yeast cells exhibitedMichaelis–Menten kinetics, as determined by the kineticdata-Wtting program EnzWtter (Fig. 6). The apparent Kmvalues, obtained using non-linear regression analysis, were38§ 10�M for glucose (Fig. 6A) and 16§5 mM for fruc-tose (Fig. 6B). These results show that TBXHT1 is a “very-high-aYnity” transporter for glucose and a “moderate-aYnity” transporter for fructose. To gain further insightinto the substrate speciWcity of TBHXT1 the uptake of glu-cose was challenged with an excess (100-fold comparedwith glucose Km) of diVerent unlabeled sugars. The insert ofFig. 7 shows the eVect of diVerent glucose analogues(DOG) on glucose uptake: 2DOG produced a level ofinhibition comparable to that caused by the addition ofunlabeled D-glucose, 6DOG and 5TG caused a minor com-petition, whereas 1DOG and 3DOG were not competitors,demonstrating that the hydroxyl groups in the C1 and C3positions are involved in the interaction with TBHXT1.T. borchii transporter shows a high aYnity also for D-man-nose and 3OMG, whereas galactose does not inhibit glu-cose uptake, showing the importance of the C4 position inthe substrate recognition (Fig. 7). The addition of the alco-hol sugars, sorbitol and mannitol, or the pentose sugars,arabinose and xylose, had no eVect on the uptake of

Fig. 4. In (A), topological model of TBHXT1 according to the consensus prediction of four prediction methods. TM helices are labeled with Romannumerals. Amino acids in bold correspond to the G-R-[KR] motifs typical of the transporters belonging to the MFS Superfamily and the arrows indicatethe other two patterns found along the protein sequence and described in the text. (B) Helix boundaries predicted by concordant prediction programs.Boundaries chosen for the TBHXT1 topology model shown in (A) are in bold.

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OUT I II III VIV VI VII VIII IX X XI XII

I II III IV V VI VII VIII IX X XITMHMM2.0 12-34 65-87 94-116 120-142 149-171 186-205 273-292 307-329 336-358 368-390 403-425

SOSUI 21-43 64-86 94-116 119-141 153-174 185-207 275-297 305-327 336-358 362-384 404-426

TMPRED 15-34 67-90 94-112 124-140 153-171 188-206 273-394 308-330 337-357 350-383 403-427

HMMTOP 15-34 65-83 96-115 120-139 152-171 186-205 273-292 307-326 339-363 368-390 403-422

A

B


D-[U-14C]glucose. This result suggests that Tbhxt1 codes fora hexose transporter and pentose and alcohol sugars enterinto the cell through other uptake mechanisms. To obtaininformation on the energization mode of the hexose trans-port we tested the glucose uptake after the addition of anATPase inhibitor or uncouplers. TBHXT1 is not sensitiveto the addition of orthovanadate, whereas the protono-phores CCCP or DNP cause a drastic reduction of D-glu-cose uptake corresponding to 95% and 70%, respectively(data not shown). These results demonstrate that TBHXT1accumulates glucose via a proton-symport mechanism.

3.4. EVect of the carbon sources and plant–fungus interaction on the Tbhxt1 expression level

A quantitative real-time PCR was set up in order tomonitor the expression of the Tbhxt1 gene in mycelia cul-tured under the growth conditions described in Section 2.Fig. 8 shows that the Tbhxt1 expression level do not changesigniWcantly when mycelia, precultivated in MMN liquid

Fig. 5. Functional complementation analysis of TBHXT1 in yeast. (A)The hxt-null S. cerevisiae strain EBYVW.4000 (1) was transformed withthe p426HXT7-6His expression vector alone (2) or with the same vectorcarrying the Tbhxt1 cDNA (3) and tested for its ability to grow on YNBmedium containing glucose or fructose at a concentration of 2% as solecarbon source. Growth was examined after 4 days at 30 °C. (B) Consump-tion of extracellular hexoses by yeast cells expressing TBHXT1. Opensymbols represent the strain EBYVW.4000 containing the vector aloneand Wlled symbols represent the same strain complemented with Tbhxt1.Glucose (�,�) and fructose (�, �) were the sugars tested. Error barsshow the SD of the triplicate results at every point in time.

1 2

3

Glucose

3

21

Fructose

0

0.5

1

1.5

0 30 60 90 120

Time (min)

Ext

ra c

ellu

lar

hex

ose

con

cent

rati

on(m

M)

A

B

medium containing glucose, are transferred for three daysto MMN medium containing fructose at concentrationsranging from 0.1 to 50 mM. On the contrary, the amount ofTbhxt1 messenger depends on the glucose concentrationutilized in the growth medium. Tbhxt1 is repressed in

Fig. 6. Kinetics of glucose (A) and fructose (B) uptake by yeast cellsexpressing TBHXT1. Michaelis–Menten plots with correspondingLineweaver–Burke plots (insets). The hexose uptake rate was calculatedfrom the radioactivity incorporated by yeast cells.

A

B

Fig. 7. In the competition experiments, the yeast strain EBYVW.4000expressing TBHXT1 was incubated (1 min at 30 °C) with 38 �M D-[14C]glucose in the presence of 100-fold excess of diVerent sugars andDOG analogues (inset). The uptake rate of D-[14C]glucose is reportedin percentage of control without competitor added and 100% uptake cor-responds to 225 pmol min¡1 108 cells¡1.

0

40

80

120

Contro

l

Gluco

se

Fructo

se

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ose

3OM

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se

Xylose

Arabin

ose

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(%)

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Contro

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1DOG

2DOG

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6DOG


mycelia shifted for three days in very low (0.1–1.0 mM) orin very high (50 mM) glucose concentrations, whereas itsexpression is weakly induced by low glucose concentrations(3 mM). Another experiment was designed to test the eVectof carbon starvation conditions. The mycelia were trans-ferred to carbon starvation condition for 3, 7 or 20 days.Fig. 8 shows that mRNA levels increase about ten foldwhen the carbon starvation was prolonged to 20 days com-pared with the mRNA level of the control. Moreover,Tbhxt1 gene expression, evaluated in our in vitro T. borchii–T. plathyphyllos ectomycorrhizae, do not change respect tothe control.

4. Discussion

Many investigations have been performed on the molec-ular genetics of hexose transport in several yeast species,such as S. cerevisiae, Kluyveromyces lactis, Schizosaccharo-myces pombe and Pichia stipitis (Boles, 2002). Nevertheless,on a molecular level, very little information is available onhexose transport in Wlamentous fungi (Schneider andWiley, 1971; vanKuyk et al., 2004) and only few transport-ers are described in plant-interacting fungi. All these genesbelong to basidiomycete fungi, such as A. muscaria (Nehlset al., 1998; Nehls, 2004), U. fabae (Voegele et al., 2001) andP. involutus (Le Quere et al., 2006).

In this paper, we report the isolation and the functionalcharacterisation of the Wrst hexose transporter gene(Tbhxt1) from the ectomycorrhizal ascomycetous fungus T.borchii. Tbhxt1 encodes for a membrane protein with a

Fig. 8. Real-time PCR quantiWcation of Tbhxt1 mRNA in ectomycorrhi-zal tissue and free-living mycelia pre-grown for 20 days in liquid MMNmedium containing 10 mM glucose and transferred for 3 days to liquidMMN medium supplemented of glucose or fructose at concentrationsranging from 0.1 to 50 mM or no carbon source (starvation) for 3, 7 and20 days. F, fructose; G, glucose; ecto, ectomycorrhiza; STV, starvation.The sugar concentration is expressed in millimolar. Tbhxt1 expression lev-els were normalised against 18S rRNA levels. Relative Tbhxt1 abundancein mycelia grown for 23 days in MMN liquid medium containing 10 mMglucose (control) is indicated by the horizontal line. The values aremeans § SE of six independent experiments.

33.23±6.5

0

1

2

3

4

5

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deduced secondary structure sharing several features withmembers of the Major Facilitators Superfamily (MFS)(Marger and Saier, 1993).

The TBHXT1 best protein sequence similarity has beenfound with the functionally characterised monosaccharidetransporters of N. crassa RCO-3 (Madi et al., 1997), A.muscaria MST1 (Nehls et al., 1998) and U. fabae HXT1(Voegele et al., 2001), but also displayed a signiWcant simi-larity throughout its entire sequence with other putativefungal hexose transporters.

Moreover, as is the case of the A. muscaria and U.fabae, low- and high-stringency Southern blots showedthat a single copy of the gene is present in T. borchiigenome. Nevertheless this result does not exclude the pos-sible existence of Tbhxt1 paralogues, as reported for A.muscaria where a second transporter gene with a low sim-ilarity to AmMst1 has been identiWed (Nehls, 2004). Onthe contrary, in other fungi, such as S. cerevisiae, 20 diVer-ent glucose transporters have been isolated, all of themwith signiWcant similarities in their sequences (Boles andHollenberg, 1997). The complex life cycle of the ectomy-corrhizal ascomycetous fungus T. borchii is characterizedby a Wrst stage as a Wlamentous mycelium (vegetativephase), a second phase of symbiotic association of thefungal hyphae with the host roots (ectomycorrhiza) andWnally the organization of a hypogeous ascomata (Talouand Kulifaj, 1992). In the vegetative phase the concentra-tion of glucose available to the fungus in the soil rangesfrom 0.1 to 1.0 mM (Wainwright, 1996). During the estab-lishment of the ectomycorrhiza, in the apoplastic com-partment of the plant–fungus interface, sucrose ishydrolysed into equimolar concentrations of glucose andfructose. A hexose gradient between the Hartig net, with asugar concentration over 2 mM, and the fungal sheath,with lower hexose concentration (below 0.5 mM), hasbeen reported (Nehls et al., 2001). The low glucose con-centration available justiWes the presence in T. borchii of ahexose transporter with a high aYnity for glucose. Uptakekinetics obtained for TBHXT1 expressed in the hxt-null S.cerevisiae strain EBYVW.4000 showed that this proteinhas a strong preference for glucose (Km V 38§ 10 �M)over fructose (Km V 16§ 5 mM). Interestingly, the glucoseKm value of TBHXT1 is about 10–12 fold lower than thatreported for MST1 of A. muscaria (Km(glucose)D 0.46 mM)(Wiese et al., 2000) or HXT1 of U. fabae(Km(glucose) D 0.36 mM) (Voegele et al., 2001). This Wndingis noteworthy because these are the only glucose trans-porters from an ectomycorrhizal fungus or a biotrophicplant pathogen fungus to be characterised until now. Fur-thermore, the Km value for fructose is about 4-fold higherthan the one reported for MST1 of A. muscaria(Km(fructose)D 4.2 mM) (Wiese et al., 2000) and 16-foldhigher than the value of U. fabae HXT1(Km(fructose)D 1.0 mM) (Voegele et al., 2001). Hence, show-ing a signiWcantly lower Km value for glucose, TBHXT1could be deWne a “very-high-aYnity” glucose transportersimilar to MSTA of A. niger (vanKuyk et al., 2004). More-


over, the aYnity of the transporter for its substrate corre-lates with the glucose concentration in the environment inwhich T. borchii lives.

The hexose uptake seems to occur by co-transport withprotons because it was completely abolished by the proton-ophores CCCP and DNP, as described in other fungi(Wiese et al., 2000; vanKuyk et al., 2004; Voegele et al.,2001).

The characterization of substrate–transporter interac-tion was studied using diVerent hexose analogues as com-petitors for glucose transport. The data reported in Fig. 7show the primarily carbon atoms involved in this process.The use of DOG analogues allowed us to evaluate the mostimportant hydrogen bonding at each carbon position inTBHXT1. The fact that 2DOG, 5TG and 6DOG reducedthe uptake of labeled glucose shows that the C2, C5 and C6positions have little inXuence on the substrate recognitionby TBHXT1. Removal of oxygen atoms from the C1 andC3 positions (1DOG and 3DOG) does not reduce the glu-cose uptake, demonstrating that hydroxyl groups in thesepositions are involved in high-aYnity interaction with theT. borchii transporter. Regarding the C3 position, the use of3OMG determined, in contrast to 3DOG, a high reductionof glucose transport showing that there is probably suY-cient space in the ligand-binding pocket of TBHXT1 toaccommodate a methyl group. D-Mannose, the C2 glucoseepimer, led to a signiWcant decrease of glucose uptakeaccording to the hypothesis that the C2 position is notinvolved in the recognition by the transporter. D-Galactose,the C4 epimer, did not show any reduction in the glucoseuptake, revealing that this transporter distinguishes pyra-noses by steric hindrance at the C4 position. Aldopentose,xylose and arabinose, lacking the C6 carboxyl group, arenot transported by TBHXT1, unlike 6DOG, suggestingthat the C6 hydrogen bonding is not suYcient for the rec-ognition by the transporter, but a steric requirement is nec-essary. Mannitol and sorbitol, analogues of the open formof glucose, did not inhibit glucose transport by TBHXT1,demonstrating that only cyclic forms bind to the trans-porter. Regarding fructose, at equilibrium in aqueous solu-tion, this sugar is present in a furanose form for 25–30%.This ketose probably binds to TBHXT1 in �-furanoseform, a virtually similar structure to �-glucopiranose.

Since T. borchii hexose transport properties should bereXected in the expression pattern of the gene, a quantita-tive real-time PCR was performed in C-starved mycelia orin mycelia supplemented of diVerent glucose or fructoseconcentrations. Tbhxt1 messenger is not regulated inmycelia shifted for three days in fructose at diVerent con-centrations; in fact the transcript shows the same level ofthe control. On the contrary, the amount of Tbhxt1 mes-senger depends on the glucose concentration utilized inthe growth medium and a negative feed-back regulationmechanism by very low and high glucose concentrationswas observed. This expression pattern is similar to thatreported in S. cerevisiae for HXT2 and HXT4 subjected toindependent repression mechanisms mediated by Rgt1

and Mig1 (Özcan and Johnston, 1999). Nevertheless, theresults reported for Tbhxt1 diVer from previous studies onA. muscaria, where high glucose and fructose concentra-tions (50 mM) trigger a 4-fold increase of the AmMst1transcript level. The same increase was observed in A.muscaria ectomycorrhizae where, after the preferentialglucose uptake, the observed enhancement of apoplasticfructose concentration is responsible of the high AmMst1gene expression (Nehls et al., 1998). On the contrary, ourinvestigations show no increase in Tbhxt1 transcript inthe ectomycorrhizal tissue. Considering the expressionpattern of Tbhxt1 gene and the kinetic characteristics ofthe encoded protein, it appears that TBHXT1 is mainlyinvolved in the hexose uptake regulation during the sap-rophytic mode of nutrition of T. borchii and not associ-ated with the symbiotic process. Nevertheless, we can notexclude the presence of additional hexose uptake systemsin the ectomycorrhizal structure.

A further evidence that Tbhxt1, unlike AmMst1, is notupregulated by high hexose concentrations lies in the factthat the amount of Tbhxt1 transcript is increased by pro-longed carbon deprivation (Fig. 8). In literature it has beenreported that high-aYnity sugar transporters are only pro-duced in the presence of low concentrations or in theabsence of preferred monosaccharides (vanKuyk et al.,2004). Tbhxt1 transcript levels in mycelia grown in glucosestarvation for 3 or 7 days are lower than the control. Thisbehaviour was observed also during yeast glucose starva-tion, where the Rgt1, bond to the HXT promoters,represses HXT2 and HXT4 expression. After 20 days ofstarvation the transcript increases of about 30-fold; thislong-term response is likely controlled in a complex mannerinvolving both a negative regulation, in the Wrst carbonstarvation phase, and a positive regulation during the pro-longed starvation. A similar delayed response was alsoobserved for the T. borchii ammonium transporterTbAMT1, which reaches its highest level of expression 21days after mycelium transfer to nitrogen-deprived medium(Montanini et al., 2002). Hence, it appears that long-termresponses aim to improve the carbon and nitrogen assimila-tion capacity of free-living mycelia before the mycorrhizalinteraction.

In conclusion, Tbhxt1 is a fully functional hexosetransporter and compared with AmMst1, the only mono-saccharide transporter from an ectomycorrhizal fungus,the three most distinctive features of Tbhxt1 are the highaYnity of the encoded-protein for glucose, a negativefeed-back regulation mechanism by very low and highglucose concentrations and its expression in the ectomy-corrhizal tissue at the same level of vegetative mycelium.These data lead us to maintain that TBHXT1 is mainlyinvolved in the very eYcient glucose uptake during T. bor-chii vegetative phase and we therefore expect the presenceof additional hexose uptake systems in the ectomycorrhi-zal structure.

We are currently testing this hypothesis by investigat-ing putative hexose transporter/s which are expressed


during the symbiotic phase to gain a complete under-standing of the hexose uptake in all the phases of T. bor-chii life cycle.

Acknowledgments

We are grateful indebted to Dr. E. Boles for gifts ofEBYVW.4000 and the expression vector p426HXT7-6Hisand for the useful information provided. We also wish tothank Dr. A. Zambonelli of the University of Bologna forkindly providing T. borchii mycelium strain MYA-1019,Dr. A. Viotti of the University of Milano for T. borchiimycelium cDNA library and Prof. Giovanna Giomaro andDr. Davide Sisti of the University of Urbino for T. borchii–T. platyphyllos ectomycorrhizae. This work was supportedby PRIN: CoWn, 2003.

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