LETTERS

Characterization of a carbohydrate transporter fromsymbiotic glomeromycotan fungiArthur Schußler1{, Holger Martin1, David Cohen1, Michael Fitz2 & Daniel Wipf2

The symbiotic relationships between mycorrhizal fungi and plantshave an enormous impact on terrestrial ecosystems1. Most commonare the arbuscular mycorrhizas, formed by fungi belonging to thephylum Glomeromycota2. Arbuscular mycorrhizal fungi facilitatethe uptake of soil nutrients by plants3 and in exchange obtain car-bohydrates, thus representing a large sink4 for atmospheric plant-fixed CO2. However, how carbohydrates are transported throughthe symbiotic interface is still unknown. Here we report the char-acterization of the first known glomeromycotan monosaccharidetransporter, GpMST1, by exploiting the unique symbiosis of aglomeromycotan fungus (Geosiphon pyriformis) with cyanobac-teria5. The GpMST1 gene has a very low GC content and containssix introns with unusual boundaries. GpMST1 possesses twelvepredicted transmembrane domains and functions as a proton co-transporter with highest affinity for glucose, then mannose, galac-tose and fructose. It belongs to an as yet uncharacterized phylogen-etic monosaccharide transporter clade. This initial characterizationof a new transporter family involved in fungal symbiosis will lead toa better understanding of carbon flows in terrestrial environments.

Members of the Glomeromycota are multikaryotic, asexual, oblig-ate symbionts, and as such render certain studies difficult or evenimpossible. We use the unique symbiosis of a glomeromycotan fun-gus (G. pyriformis) with a cyanobacterium (Nostoc punctiforme) toget a handle on the role of fungal transcripts in carbohydrate trans-port4. This symbiosis is highly interesting because (1) it is the onlyknown fungal endosymbiosis with cyanobacteria and (2) the sym-biotic stage (that is, the ‘bladder’) is comparable to the within-rootsituation of the arbuscular mycorrhiza5,6 (Fig. 1). The bonus for geneexpression studies is that, contrary to ‘real’ arbuscular mycorrhizas,fungal messenger RNA (mRNA) can be isolated specifically from thesymbiotic stage.

To characterize the first glomeromycotan sugar transporter wefunctionally complemented the hexose-uptake deficient yeast strain7

EBY.VW4000 with a complementary DNA (cDNA) expression lib-rary established from symbiotic G. pyriformis bladders. Screeningsresulted in yeast clones containing plasmids with a cDNA insert of2,043 bp (Fig. 2a). The cDNA encodes an ORF of 540 amino acids,which contains the PROSITE sugar transport proteins signature2, PS00217 (http://www.expasy.org/prosite/PS00217; consensuspattern [LIVMF]xG[LIVMFA]{V}xG{KP}-x(7)-[LIFY]-x(2)-[EQ]-x(6)-[RK]), and the sugar transporter PFAM motif, PF00083(http://www.sanger.ac.uk//cgi-bin/Pfam/getacc?PF00083). The genewas shown (see below) to code for a monosaccharide transporter(MST) and named GpMST1. It codes for a 12 transmembranedomain (TMD) topology8 transporter of the major facilitator super-family with intracellular termini, a large extracellular loop (betweenTMD1 and TMD2) that is shared by most hexose transporters9, and arelatively long10 central loop (Fig. 2b).

Phylogenetic analyses were performed to discover to which of theknown MST clades GpMST1 belongs (Supplementary Figs 1, 2).Results of BLASTP searches comprised only 10–14 fungal sequenceswithin the 200 best hits, with highest identities/similarities of 40/60%,respectively, to animal and fungal sequences. Phylogenetic analysesof 1,620 transporters revealed that GpMST1 does not cluster withinany of the previously defined MST clades10, but within a distant fun-gal clade. The clade consists of (mostly from recent genome pro-jects) sequences from 16 ascomycete fungi (Saccharomyces cerevisiaeQ6B2Y5; YB91_YEAST; VPS73_YEAST; Kluyveromyces lactis Q8J289;Candida glabrata Q6FWZ2; Candida albicans Q59UD9 and Q5ALJ1;Debaryomyces hansenii Q6BY36 and Q6BJ92; Yarrowia lipolyticaQ6CDU0; Ashbya gossypii Q754V5; Aspergillus fumigatus Q4WYR5;Aspergillus oryzae Q2UC56; Gibberella zeae Q4IE71; Blumeria grami-nis Q8NK49; and Neurospora crassa Q870X7) and 3 basidiomycetefungi (Cryptococcus neoformans Q55TK8 and Q55VM4; and Ustilagomaydis Q4PE10). GpMST1 is presumably the first characterizedtransporter of this new and interesting clade of fungal MSTs.However, because the phylogenetic distance between dikaryotic fungi(Ascomycota and Basidiomycota) and Glomeromycota is very large(putative split .700 Myr ago) the clade is not strongly supported andmore glomeromycotan sequences will be needed before drawing anyfinal conclusions.

1Darmstadt University of Technology, Institute of Botany, Schnittspahnstrasse 10, 64287 Darmstadt, Germany. 2University of Bonn, IZMB, NWG Transport in Mycorrhiza, Kirschallee1, 53115 Bonn, Germany. {Present address: Bereich Genetik, Department Biologie I, Maria-Ward-Straße 1a, 80638 Munchen, Germany.

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Figure 1 | Comparison of bidirectional phosphate and carbon exchanges.Despite inverted relative dimensions of macro- and microsymbiont theinterface and nutrient exchange in the G. pyriformis symbiosis correspond tothat in the arbuscular mycorrhiza. Several arbuscular mycorrhiza-specificphosphate transporters (PT) are known from plants. The hypothetical roleof GpMST1, and its orthologues, in the sugar uptake through the symbioticmembrane of glomeromycotan fungi is indicated together with thesubstrates of GpMST1 (fructose and putatively xylose are transportedweakly).

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The GpMST1 gene is 2,678 bp long and contains 6 introns (Fig. 2a)and 3 functional polyadenylation signals (cDNA with poly(A) tailsbeginning after all three signals were sequenced). A typical feature ofarbuscular mycorrhizal fungi (AMF) genomes seems to be an excep-tionally low GC content. For example, Glomus intraradices has anaverage of 36.3% GC among its codons11, and the same holds true forGpMST1 (codons 37.6%; genomic DNA 31.4%; introns 22%). Theintron–exon boundaries of the six introns (Supplementary Fig. 3) aresignificantly influenced by the low GC content. Half of them containan A as the first exon-nucleotide at the 39 splicing site, and, unusually,two have the pyrimidine T. At the third position upstream of the 39

splicing site .90% of human and yeast12 introns contain a pyrimi-dine (T or C), but two-thirds of the GpMST1 introns show a purine(A) base. Such unusual features may be expected for other glomer-omycotan genes, too. To reveal whether GpMST1 is expressed in aphotosynthesis-dependent manner, its expression after 47 or 70 h ofdarkness, or after 18, 47 or 70 h of light was studied by semi-quant-itative RT–PCR (PCR after reverse transcription): the gene seems tobe constitutively expressed over this time (data not shown).

To investigate the substrate specificity, the GpMST1 expressingyeast mutant EBY.VW4000 was plated on sugars and sugar derivates.The clones could not grow on D-sucrose, D-lactose, D-cellobiose,D-trehalose, D-arabinose, L-arabinose, D-ribose, D-xylose myo-inositol, D-sorbitol, D-glucosamine N-acetyl-glucosamin, L-glucose,L-rhamnose and L-fucose. Growth was reconstituted on D-fructose(very weak), D-galactose (moderate), D-mannose (high) andD-glucose (highest). To investigate whether growth rates were dueto the affinity of the transporter or to altered yeast metabolism,competition experiments were performed (Fig. 3a). Xylose was alsotested because it is a main constituent of plant cell walls and somefungal MSTs transport xylose at low rates13,14; indeed, this was alsoindicated for GpMST1. Uptake of 14C-glucose (at pH 6.5) throughGpMST1 was very sensitive to protonophores and H1-ATPase inhi-bitors (Fig. 3b), indicating secondary active H1 co-transport. Theglucose uptake was pH-dependent, with a somewhat surprisingoptimum of pH 7 (Fig. 3c). We used pH 6.5 for all experiments toensure working with a DpH . 0.5, and because most yeast sugar

transport studies were performed at this pH. Michaelis–Menten kin-etics and Lineweaver–Burk transformation indicate a Vmax of,0.18 nmol glucose per 106 cells per min and a KM of ,1.2 mMfor glucose uptake (Fig. 3d). GpMST1 therefore represents the firstknown glomeromycotan sugar transporter, transporting monosac-charides with rates in the order glucose.mannose.galactose.

fructose.Many symbiotic Nostoc strains seem to be capable of heterotrophic

growth on sucrose, glucose or fructose, such as the strain PCC73102—which forms symbioses with Anthoceros and Gunnera15—and also the symbiont of G. pyriformis16. However, contrary to theplant symbioses formed with Anthoceros and Gunnera, carbohydratetransport in the G. pyriformis symbiosis is in the opposite direction,from photoautotrophic symbiont to fungus, as in the arbuscularmycorrhiza.

There are some indications that GpMST1 transports hexosesacross the symbiotic interface membrane. On the one hand, whenexpressed in yeast, it is clearly active in the plasma membrane; on theother hand, for G. pyriformis incubated in 14C-glucose no uptakefrom the outside could be shown (M. Kluge, personal communica-tion), although metabolites originating from cyanobacterial 14CO2

fixation could be easily detected17. The G. pyriformis membrane at thesymbiotic interface is derived from the plasma membrane (Fig. 1),and a chitin-containing ‘cell wall’ layer, which is similar to the arbus-cule cell wall, is synthesized within the bladders18. Our interpretationis that GpMST1 probably represents the type of MST that is respons-ible for the transport of plant carbohydrates to glomeromycotanfungi through the symbiotic membrane, and that G. pyriformis, aswell as ‘typical’ AMF4,19, are not able to take up glucose through thenon-symbiotic plasma membranes.

Carbon flow is a key process in the arbuscular mycorrhiza, andsucrose is usually interpreted to be the source of hexoses taken up byAMF. The N. punctiforme symbiont of G. pyriformis synthesizes suc-rose15 for osmoregulation, which therefore could also be a C sourcefor the fungus. However, another aspect might be interesting: besidesglucose, GpMST1 efficiently transports main ‘cell wall hexoses’—mannose and galactose. This unusual substrate specificity led us to

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the speculation that at least a portion of the monosaccharides couldbe liberated from plant extracellular polysaccharides and taken up asa C source by AMF, as indicated in Fig. 1. In the G. pyriformis sym-biosis the extracellular polysaccharides synthesized by N. punctiformeseem to be degraded. In arbuscular mycorrhizas, the plant ‘cell wall’in the interface space is also very thin and was shown to be amorph-ous and to contain mainly glucose, mannose, galactose, xylose, andarabinose20. Recently, high activities of xyloglucanases and endocel-lulases were reported in arbuscular mycorrhizal roots21,22, and it wasshown that the growth of AMF is significantly enhanced by certainsugars that are not derived from sucrose cleavage, for example, raffi-nose and other trisaccharides23. Might extracellular polysaccharidesbe an alternative energy source for AMF? Could the plant vesicletransport towards the fast-growing perisymbiotic membrane beinvolved in C-supply and regulation of root colonisation?

There are many open questions, but the glomeromycotan MSTcharacterized here should boost this field by enabling the isolationand characterization of orthologues, which will allow expressionprofiling in arbuscular mycorrhizas. This may lead, in the near future,to a much better understanding of arbuscular mycorrhizas and therelated global carbon fluxes.

METHODSOrganisms. The G. pyriformis–N. punctiforme symbiosis was grown in our labor-

atory as described5. S. cerevisiae strains used were the hxt-null mutant

EBY.VW4000 and its parental strain CEN.PK2-1C (ref. 7).

Messenger RNA isolation, reverse transcription and cDNA amplification.

Five-hundred G. pyriformis bladders were frozen in liquid N2 within a 2 ml vial

containing a tungsten carbide bead. Deep-frozen samples were ground in a bead

mill (MM2000, Retsch) and ,80 ng of poly(A)-mRNA was isolated with mag-

netic oligo(dT)-beads (Dynabeads mRNA DIRECT kit, Dynal Biotech). Reversetranscription was performed and double-stranded cDNA amplified with the

Creator SMART ds-cDNA synthesis kit (Clontech, BD Biosciences).

Construction of the cDNA library. Amplified cDNA was size-fractionated

(.1.1 kb) and concentrated. Five-hundred ng was digested with Sfi I and ligated

into pDR196 vector24, which was modified to contain asymetric Sfi I sites.

pDR196sfi–cDNA plasmids were then agarose-gel-extracted and transfected into

Escherichia coli XL 10 Gold (Stratagene). Clones (6 3 105) were harvested; ,95%

were recombinant and average insert size was ,1.1 kb.

Isolation of GpMST1 by functional complementation. Clones (105) were har-

vested from the primary library. After transformation25 of EBY.VW4000, selec-

tion was performed on synthetic dextrose (SD) agar (without uracil) containing

2% maltose instead of glucose. Colonies were harvested and aliquots plated on

SD agar (without uracil) containing 22 mM glucose as the C source. pHM13-C6

(representative of several clones) was isolated, transfected into E. coli, re-isolated,

and re-transfected into EBY.VW4000 for further characterization of three sub-

clones.

Genomic DNA sequencing and amplification. Genomic DNA (1 ng) from G.

pyriformis was amplified with the Phi29-DNA- polymerase-based GenomiPhi

DNA amplification kit (GE Healthcare), resulting in .5 mg of amplified gDNA.

Primers designed for the termini of cDNA sequences were used to amplify

the GpMST1 gene with the Phusion proof-reading polymerase (Finnzymes).

The full-length fragment (pDC13-C6) was cloned in pCR4Blunt-TOPO

(Invitrogen) and sequenced, as well as a further eight overlapping fragments

from three independent gDNA extractions.

Semi-quantitative RT–PCR. G. pyriformis bladders were stochastically distrib-

uted (5 Petri dishes, 50 bladders each) and subjected to 18, 47 or 70 h of light, or

47 or 70 h darkness. Bladders were collected in RNase-free vials, immediately

frozen in liquid N2, and mRNA was isolated using Dynabeads, but without heat

elution. The beads–mRNA complex was resuspended in reverse transcription

mixture (Sensiscript RT kit, QIAGEN, Hilden). Reverse transcription was per-

formed using the oligo(dT)-beads as primer, resulting in covalently attachedcDNA. A 720 bp GpMST1 and a 520 bp EF1a cDNA fragment (specificity

checked by sequencing) were sequentially amplified by solid-phase PCR with

Phusion proof-reading polymerase. Samples were taken after 15, 19, 23, 27 and

31 cycles and analysed by agarose gel electrophoresis.14C-glucose uptake studies. Yeast cells were grown to A600 nm 0.5, harvested,

washed in water twice and resuspended in buffer A (0.6 M sorbitol, 50 mM

potassium phosphate) to A600 nm 5.0. Before measurements, cells were incubated

in 8 mM maltose for 5 min. The reaction was started by adding 100ml of cell

suspension to 100ml of buffer A containing at least 7.4 kBq of 14C-glucose

(Amersham Biosciences) and unlabelled glucose at the concentrations used in

the experiments. After 30, 60, 120 and 240 s, 50 ml aliquots were transferred to

4 ml of ice-cold buffer A, filtered on glass fibre filters, and washed twice with

buffer before liquid scintillation spectrometry. Competition for glucose uptake

was performed by adding a fivefold molar excess (5.5 mM) of the respective

competitors to 1.1 mM glucose. pH dependence was analysed in 100 mM pot-

assium phosphate buffer adjusted to different pH values. The influence of the

electrochemical gradient was analysed by incubation (5 min) in 1.1 mM glucose

(control), or in glucose and 2,4-dinitrophenol (DNP), diethylstilbestrol (DES),

carbonyl cyanide m-chlorophenylhydrazone (CCCP) or vanadate (100mM each).

All measurements were repeated independently in at least three experiments.

Phylogenetic analysis. The GpMST1 amino acid sequence was first aligned with

the 200 best BLASTP hits. From the alignment, 251 sites could be used. Identical

sequences were removed and 170 sequences analysed with a quartet puzzling

maximum likelihood method (TREE-PUZZLE 5.2). Frequencies of amino acids

were estimated from the data set, rate heterogeneity taken into account (invari-

able and gamma-distributed rates), and WAG, JTT and VT models used. Further

analyses used the transporter alignment (2,174 sequences) from http://www.

sanger.ac.uk/cgi-bin/Pfam/getacc?PF00083. After removing short sequences

the data set was composed of 1,620 sequences (375 sites) and the fungal data

set of 742 sequences (380 sites). The 1,620 sequences were used to construct

distance and parsimony trees with PROTDIST and PROTPARS (PHYLIP 3.6;

see Supplementary Figs 1 and 2).

Received 9 July; accepted 20 October 2006.

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Supplementary Information is linked to the online version of the paper atwww.nature.com/nature.

Acknowledgements We thank E. Boles (University of Frankfurt) for providingEBY.VW4000 and CEN.PK2-1C yeast strains, D. Rentsch (University of Bern) forproviding pDR196, and J. Gossmann (University of Frankfurt) for proofreading. Thiswork was supported by grants from the Deutsche Forschungsgemeinschaft to D.C.,H.M. and A.S.

Author Contributions A.S., H.M. and D.C. isolated the full-length GpMST1 cDNAclones and performed semiquantitative RT–PCR. A.S. and D.C. retransformedEBY.VW4000, and performed screenings and growth tests. H.M. modified thepDR196 vector, constructed the cDNA libraries, and performed initial yeasttransformations and screenings. M.F. performed the uptake assays. D.C. helpedwith uptake assays and performed gDNA amplification, PCR and clonings. A.S.made the phylogenetic analyses; A.S. and D.W. are responsible for theexperimental design, hypotheses, interpreting the results and writing themanuscript.

Author Information Sequences are deposited at EMBL Data Bank with theaccession numbers AM231332 (GpMST1 cDNA clone pHM13-C6.1) and AM231333(GpMST1 gDNA clone pDC-C6). Plasmids and clones are available on request.Reprints and permissions information is available at www.nature.com/reprints. Theauthors declare no competing financial interests. Correspondence and requests formaterials should be addressed to A.S. (arthur.schuessler@lrz.uni-muenchen.de).

LETTERS NATURE | Vol 444 | 14 December 2006

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