Diversity and evolution of ABC proteins in mycorrhiza ...

RESEARCH ARTICLE Open Access

Diversity and evolution of ABC proteins inmycorrhiza-forming fungiAndriy Kovalchuk1*, Annegret Kohler2, Francis Martin2 and Fred O. Asiegbu1

Abstract

Background: Transporter proteins are predicted to have an important role in the mycorrhizal symbiosis, due to thefact that this type of an interaction between plants and fungi requires a continuous nutrient and signalling exchange.ABC transporters are one of the large groups of transporter proteins found both in plants and in fungi. The crucial roleof plant ABC transporters in the formation of the mycorrhizal symbiosis has been demonstrated recently. Some of thefungal ABC transporter-encoding genes are also induced during the mycorrhiza formation. However, no experimentalevidences of the direct involvement of fungal ABC transporters in this process are available so far. To facilitatethe identification of fungal ABC proteins with a potential role in the establishment of the mycorrhizal symbiosis, we haveperformed an inventory of the ABC protein-encoding genes in the genomes of 25 species of mycorrhiza-forming fungi.

Results: We have identified, manually annotated and curated more than 1300 gene models of putative ABC protein-encoding genes. Out of those, more than 1000 models are predicted to encode functional proteins, whereas about 300models represent gene fragments or putative pseudogenes. We have also performed the phylogenetic analysis of theidentified sequences. The sets of ABC proteins in the mycorrhiza-forming species were compared to the relatedsaprotrophic or plant-pathogenic fungal species. Our results demonstrate the high diversity of ABC genes in thegenomes of mycorrhiza-forming fungi. Via comparison of transcriptomics data from different species, we haveidentified candidate groups of ABC transporters that might have a role in the process of the mycorrhiza formation.

Conclusions: Results of our inventory will facilitate the identification of fungal transporters with a role in the mycorrhizaformation. We also provide the first data on ABC protein-coding genes for the phylum Glomeromycota and for ordersPezizales, Atheliales, Cantharellales and Sebacinales, contributing to the better knowledge of the diversity of this proteinfamily within the fungal kingdom.

Keywords: ABC transporters, Fungi, Mycorrhiza, Membrane transport, Protein family evolution

BackgroundThe family of ATP-binding cassette (ABC) proteins en-compasses a large and diverse assemblage of proteins thatare ubiquitously present in all living organisms [1]. Astheir name suggests, their principal common feature is thepresence of an ATP-binding domain. The domain providesthe energy required by ABC proteins to perform their bio-logical functions by hydrolysing ATP. A large fraction ofABC proteins, known as ABC transporters, are implicatedin transport of diverse substrates across biological mem-branes (Table 1) [2–4]. Soluble ABC proteins are not in-volved in transmembrane transport, but they play an

essential role in vital cellular processes, e.g. ribosomebiogenesis and mRNA translation (Table 1) [5]. Mostof our current knowledge is based on the results ofcharacterization of ABC transporters in two yeast species,baker’s yeast S. cerevisiae and fission yeast Schizosaccharo-myces pombe. Those data were of enormous importancefor understanding of the biological significance of ABCtransporters, in general. However, both mentioned speciesare characterised by reduced sets of ABC transporters, lack-ing many of groups that are otherwise widespread amongfilamentous fungi. Existing information on function of ABCtransporters in filamentous ascomycetes is very scarce andfragmentary, with most of the experiments being designedto assess the role of individual transporters in drug resist-ance, whereas ABC transporters of basidiomycetes have

* Correspondence: [email protected] of Forest Sciences, University of Helsinki, P.O. Box 27, FIN-00014Helsinki, FinlandFull list of author information is available at the end of the article

© 2015 Kovalchuk et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Kovalchuk et al. BMC Evolutionary Biology (2015) 15:249 DOI 10.1186/s12862-015-0526-7

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almost completely escaped the attention of experimentalscientists.Numerous species of fungi are known to form mycor-

rhizae, symbiotic interactions between fungi and higherplants. It is estimated that more than 90 % of all plantspecies, including forest trees, wild grasses and manycrops are participating in mycorrhizal interactions [6].Mycorrhiza is a rather broad term that describes structur-ally heterogeneous types of interactions between fungi androots of higher plants. In case of ectomycorrhiza, fungalhyphae surround the root tips and can grow between

epidermal cells, but never enter cell lumen. On contrary,endomycorrhiza-forming fungi penetrate inside living cellsof the root epidermis and cortex [6]. Endomycorrhizae canbe further subdivided into ericoid, arbutoid, orchid andarbuscular mycorrhiza. Mycorrhiza-forming fungi are alsovery diverse taxonomically, as they represent different evo-lutionary lineages from three fungal phyla, Glomeromy-cota, Ascomycota and Basidiomycota. Phylogenetic analysisindicates that mycorrhizal symbionts have evolved in-dependently in different lineages of fungi [7]. Establish-ment of mycorrhizal interaction is a complex process

Table 1 Biological functions of different groups of fungal ABC proteins

Group Function References

ABC-A Biological function in fungi unknown, but Magnaporthe grisea transporterAbc4 is required for appresoria formation. Mammalian ABC-A transportersare involved in lipid transport and metabolism

[29–31]

ABC-B full ABCB1 Export of mating pheromones [32–34]

ABCB2 Multidrug resistance; a specific group of ABCB transporters in ascomycetes(but not in Agaricomycotina) is associated with the biosynthesis cluster forthe hydroxamate-type siderophores and might have a role in their metabolism.

[14, 35–37]

half ABCB3 Export of the mitochondria-synthesized precursors of iron-sulfur (Fe/S) clusters;localised to the inner membrane of mitochondria

[38, 39]

ABCB4 Heavy-metal resistance; localized to the vacuolar membrane [40–42]

ABCB5 Transporter of the inner mitochondrial membrane involved in the export ofpeptides released upon proteolysis by m-AAA protease

[43, 44]

ABCB6 Unknown

ABC-C ABCC1 Vacuolar import of phosphatidylcholine; bile acid transport [45, 46]

ABCC2 Unknown

ABCC3 Unknown

ABCC4 Unknown

ABCC5 Unknown; often associated with the secondary metabolism gene clusters [14, 15, 36]

ABCC6 Heavy metal resistance; vacuolar uptake of glutathione conjugates;localized to vacuolar membrane

[42, 47–49]

ABCC7 Drug resistance; heavy metal resistance [50–52]

ABC-D ABCD1 Import of long-chain fatty acids into peroxisomes [53]

ABCD2 Import of long-chain fatty acids into peroxisomes [53]

ABCD3/4 Unknown

ABC-E Ribosome biogenesis and assembly of translational pre-initiation complexes [54, 55]

ABC-F ABCF1 Biogenesis of 40S and 60S ribosomal subunits [56]

ABCF2 Regulation of mRNA translation; positive regulator of Gcn2p kinase activity [57]

ABCF3 Unknown

ABCF4 mRNA export factor [58]

ABCF5 Translation elongation factor [59, 60]

ABC-G ABCG1-5 Pleiotropic drug resistance; weak organic acid tolerace; export of toxic metabolites,sterol uptake, translocation of membrane phospholipids and quorum sensing

[37, 61]

ABCG6 Unknown

ABCG7 Unknown; are characterised by a presence of EGF domain [14, 15]

ABC-I ABCI1 Part of the CCR4-NOT transcriptional regulatory complex [62]

ABCI2 Unknown

ABCI3 Unknown

Kovalchuk et al. BMC Evolutionary Biology (2015) 15:249 of 19

that requires a continuous nutrient and signal exchangebetween the partners [6]. Transporter proteins are ex-pected to play a key role in this process. Details of thoseinteractions are gradually being elucidated, but many ofthe involved molecular components remain unknown. Anumber of works on different plant models have shownthat specific plant ABC transporters are required for theformation of arbuscular mycorrhiza, a widespread type ofmutualistic interaction between plants and fungi of thephylum Glomeromycota [8–10], and, furthermore, it wasdemonstrated that the corresponding transporter acts asstrigolactone exporter in petunia [10]. It is not known yetwhether any of fungal ABC transporters participate in theformation of mycorrhizal interactions.To provide insight into the diversity of mechanisms

for the mycorrhizal symbiosis, the Mycorrhizal GenomicInitiative (MGI) consortium [11] performed the sequen-cing and analysis of a phylogenetically and ecologicallydiverse set of mycorrhizal fungal genomes. The set ofspecies sequenced by MGI consortium includes fungiengaged in the various types of mycorrhizal symbioticinteractions (e.g., arbuscular, ericoid, orchid mycorrhizaeand ectomycorrhiza). The species selected by MGI con-sortium belong to the phyla Glomeromycota, Ascomy-cota and Basidiomycota. They represent three differentclasses of Ascomycota (Leotiomycotina, Dothideomyco-tina and Pezizomycotina), and five orders within theclass of Agaricomycotina (Basidiomycota) (Table 2). Thegenerated data provide an excellent opportunity for thecomparative genomics analysis of evolutionary adapta-tions of fungi to the symbiosis with plants.We were interested in how the fungal ABC genes, and,

in particular, ABC transporters were influenced by theadaptations of fungi to their symbiotic lifestyle. Theseadaptations have evolved independently in different line-ages of mycorrhiza-forming fungi [12], and they show aremarkable degree of evolutionary convergence. If ABCtransporters do play a role in the process of mycorrhiza for-mation, their recruitment also occurred independently, andwe might expect to find different transporters in differentlineages depending on the type of mycorrhizal symbiosis.We would also expect to see the lineage-specific differenceswhen comparing ABC transporter repertoire betweenmycorrhiza-forming fungi and their non-symbiotic rela-tives. Therefore, when possible, we have compared thesets of ABC genes found in the genomes of mycorrhiza-forming fungi, with the situation in related saprotrophic orplant-pathogenic species, paying attention to both quanti-tative and qualitative differences in the compositionof ABC genes.We have carried out a comprehensive analysis of ABC

protein-encoding genes in the 25 fungal genomes that weresequenced by the MGI [12]. We have identified all pre-dicted ABC genes and gene fragments, manually curated

and annotated the gene models and classified them intothe subfamilies according to the HUGO-proposed scheme[13]. The identified genes were further subdivided intosmaller groups defined in our previous analyses [14, 15].We have also analysed the data on expression of ABCtransporter-encoding genes provided by members of theMGI consortium in order to identify those genes thatmight play a role in the establishment of mycorrhizal sym-biosis. We could not identify any group of ABC trans-porters that would be unique for mycorrhiza-formingfungi. However, functions of many of identified genes areunknown, and it is possible that some of them play a cer-tain role in the establishment of mycorrhizal interactions.Our data provide an insight into the diversity of ABC pro-teins in the newly sequenced genomes of mycorrhiza-forming fungi. Currently, it is not clear whether any fungalABC transporters are implicated in the mycorrhiza forma-tion. Results of our surveyshould provide a support forfurther studies on the possible role of fungal ABC trans-porters in the establishment of mycorrhizal symbiosis.Additionally, they will contribute to the better under-standing of the evolution and dynamics of this proteinfamily in the fungal kingdom.

ResultsIn the course of our inventory of ABC protein-encodinggenes in the genomes of mycorrhiza-forming fungi, wehave identified and manually curated and annotated morethan 1300 gene models from 25 fungal species (Table 2and Additional file 1; Additional file 2). 1022 of the ana-lysed gene models are predicted to encode functional pro-teins. The manual curation of the identified genes hasshown that 289 automatically generated gene models canbe further improved (Additional file 2). The most commonproblems associated with the existing gene models werethe positions of exon-intron boundaries and start and stopcodons, but we have also encountered other kinds of er-rors, e.g. missing exons or cases when a single gene hasbeen split into several (two or even three) models. Thenumber of ABC genes in the analysed genomes varies to agreat extent, from 29 in Terfezia boudieri to 72 in Oidio-dendron majus (Table 2). Subsequently, we have recon-structed the evolutionary relationships of the identifiedABC proteins (Figs. 1, 2, 3, 4, 5; Additional files 3, 4 and 5).Results of the phylogenetic analysis were in good agree-ment with our previous data [14, 15], as we could identifythe same principal groups of fungal ABC proteins. In mosttrees ABC proteins from Basidiomycota and Ascomycota,respectively, were placed in separate and well supportedmonophyletic clades. A noticeable exception is the situ-ation within the subfamily ABC-G, were the phylogeneticpattern is less clear (Additional file 5). The relationshipbetween the groups ABCG1, ABCG2, ABCG3, ABCG4,and ABCG5 is not well resolved, and their positions on


Table 2 Distribution of subfamilies of ABC proteins among analysed species of mycorrhiza-forming fungi

Species Phylum Class Order ABC-A ABC-B full ABC-B half ABC-C ABC-D ABC-E ABC-F ABC-G Others Total

Rhizophagus irregularisDAOM 181602

Glomeromycota Glomeromycotina Glomerales 0 8(9)a 3 9(11) 4(5) 1 3 4(15) 2 34(49)

Meliniomyces bicolor E Ascomycota Leotiomycotina Helotiales 3 10(13) 5 12(19) 2 1 5 13 2 53(63)

Meliniomyces variabilis F 3 9 5 26(28) 2 1 5 15 2 68(70)

Oidiodendron majus Zn 4 8(10) 7 25(26) 2 1 5 18 2 72(75)

Cenococcum geophilum 1.58 Ascomycota Dothideomycotina incertae sedis 1(3) 5(7) 4 8 2 1 5 7 2 35(39)

Tuber melanosporum Mel28 Ascomycota Pezizomycotina Pezizales 1 5 4 4 2 1 5 8 2 32

Choiromyces venosus 120613-1 1 5 4 4 2 1 5 8(11) 3 33(36)

Terfezia boudieri S1 1 4 5 5 2 1 4 5(6) 2 29(30)

Amanita muscaria Koide Basidiomycota Agaricomycotina Agaricales 1 6(8) 5(7) 9(10) 2(4) 1 3 4(11) 2 33(47)

Cortinarius glaucopusAT 2004 276

1 2(9) 8(10) 21(33) 0 1(3) 6(14) 5(9) 3 47(82)

Hebeloma cylindrosporum h7 3 3 7 13(14) 2 1 5 7 3 44(45)

Laccaria amethystina LaAM-08-1 1(2) 5(17) 7(10) 7(29) 2(4) 1 5(6) 6(13) 3(8) 37(90)

Laccaria bicolor S238N-H82 1 5(11) 7 8(14) 2 1 5 8(17) 6(8) 43(66)

Tricholoma matsutake 945 1 2(8) 12(13) 10(23) 2 1 5 8(13) 3 44(69)

Boletus edulis Basidiomycota Agaricomycotina Boletales 1 2(3) 7 6(13) 2 1 4 4(9) 3(4) 30(44)

Paxillus involutus ATCC 200175 1 4(10) 7(8) 8(14) 2 1 5 8(12) 3 39(56)

Paxillus rubicundus Ve08.2 h10 1 3(4) 7(8) 6(8) 2 1 5 7(8) 3 35(40)

Pisolithus microcarpus 441 0 5(17) 6 7(11) 3 1 3(7) 4(5) 3 32(53)

Pisolithus tinctorius Marx 270 0 5(6) 6(8) 16(18) 2 1 3 6 3 42(47)

Scleroderma citrinum Foug A 0 6 10(11) 10(13) 2 1 3 3 3 38(42)

Suillus luteus UH-Slu-Lm8-n1 1(5) 4(6) 11(13) 7(8) 2(3) 1 5(7) 6(8) 3 40(54)

Piloderma croceum F 1598 Basidiomycota Agaricomycotina Atheliales 2 7(9) 9 9(16) 2 1 5 10(16) 3 48(63)

Tulasnella calospora AL13/4D Basidiomycota Agaricomycotina Cantharellales 1 7 7 9(11) 2 1 5(6) 5(6) 3 40(44)

Piriformospora indica DSM 11827 Basidiomycota Agaricomycotina Sebacinales 1 3(4) 6 11(13) 2 1 4 4 3 35(38)

Sebacina vermifera MAFF 305830 1 6 6 13(14) 2 1 4 3 3 39(40)anumbers in parentheses include putative pseudogenes and partial sequences

Kovalchuket

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Table 3 List of ABC-protein encoding genes significantly up-regulated in mycorrhiza-forming mycelium (MYC) as compared to free-living mycelium (FLM)

Species Protein model Subfamily Gene name Fold change p-value Mean FLM Mean MYC

Laccaria bicolor 601729 ABC-B (FL) LacbiABCB2.1b 6.08 0 1097 6671

671922 ABC-B (HT) LacbiABCB6.1 36.94 1.82E-14 957 35334

327599 Ydr061w LacbiABCI2.1a 7.89 1.50E-05 328 2590

298087 Ydr061w LacbiABCI2.1d 5.90 0 504 2972

Oidiodendron maius 101847 ABC-A OidmaABCA1.2 3.66 1.35E-05 5.5 20.1

104750 ABC-B (FL) OidmaABCB2.3a 3.60 3.02E-32 35.0 125.9

101864 ABC-C OidmaABCC3.1 2.51 9.82E-05 14.5 36.5

106945 ABC-C OidmaABCC5.1 m 14.29 1.49E-12 5.1 72.5

173820 ABC-C OidmaABCC5.1n 8.32 0.029 1.0 8.2

102203 ABC-C OidmaABCC7.1a 2.11 0.004 13.7 29.0

119877 ABC-G OidmaABCG6.2a 73.40 3.02E-20 0.5 33.9

123273 ABC-G OidmaABCG6.2b 15.31 6.58E-20 6.2 95.5

Sebacina vermispora 326231 ABC-B (HT) SebveABCB6.1a 2.63 5.24E-05 13.2 34.6

19794 ABC-B (HT) SebveABCB6.1b 2.42 9.07E-05 11.5 27.9

127786 ABC-B (HT) SebveABCB6.3 6.44 8.34E-05 7.2 46.3

116826 ABC-C SebveABCC3.2e 2.06 0.002 13.6 28.1

29919 ABC-C SebveABCC7.3 3.79 2.17E-13 12.8 48.7

71362 ABC-F SebveABCF1.1 2.39 5.06E-06 66.3 158.9

333124 ABC-F SebveABCF2.1 2.68 3.60E-13 26.6 71.2

17618 ABC-G SebveABCG6.1 2.98 5.32E-06 35.4 105.7

174708 Ydr061w SebveABCI2.1 2.87 1.08E-04 10.0 28.6

69180 Ydr061w SebveABCI3.1 2.03 4.37E-04 39.1 79.5

Tulasnella calospora 19492 ABC-B (FL) TulcaABCB2.1a 2.11 2.91E-05 27.7 58.5

207894 ABC-B (FL) TulcaABCB2.1d 2.46 1.80E-04 32.4 79.6

161653 ABC-B (FL) TulcaABCB2.1e 2.43 0.001 18.4 44.8

22503 ABC-B (HT) TulcaABCB6.1a 2.13 0.014 7.4 15.8

78312 ABC-B (HT) TulcaABCB6.1b 3.75 6.96E-04 8.0 30.0

229331 ABC-B (HT) TulcaABCB6.3b 2.84 0 37.5 106.4

3597 ABC-C TulcaABCC6.1 4.39 0.002 20.1 88.1

22078 ABC-C TulcaABCC7.2 4.99 1.85E-04 4.4 22.1

72157 ABC-F TulcaABCF5.1 58.44 0.021 5.2 302.4

80372 ABC-G TulcaABCG4.1b 5.72 5.80E-06 17.4 99.3

69109 Ydr061w TulcaABCI2.1 4.67 4.32E-05 4.0 18.6

243146 Ydr061w TulcaABCI3.1 3.56 4.77E-09 14.3 50.9

Suillus luteus 17399 ABC-A SuiluABCA1.1 4.16 4.23E-10 10.5 43.7

621910 ABC-A SuiluABCA1.1 3.51 5.79E-05 6.5 22.8

797578 ABC-B (FL) SuiluABCB2.1a 2.12 7.14E-08 34.6 73.4

107869 ABC-F SuiluABCF3.1 2.28 1.08E-05 19.8 45.2

Paxillus involutus 111294 ABC-B (FL) PaxinABCB2.1c 4.21 5.22E-05 6.0 25.2

80655 ABC-G PaxinABCG2.1e 11.01 0.004 10.2 112.1

117609 ABC-G PaxinABCG2.1f 4.68 4.59E-07 7.5 35.0

Piloderma croceum 99185 ABC-B (FL) PilcrABCB2.1e 3.63 1.06E-46 52.2 189.1

4675 ABC-B (HT) PilcrABCB6.2a 3.20 1.61E-06 9.0 28.7


the tree vary depending on the species sampled for theanalysis. In addition, there are some small clades that can-not be assigned to any of the larger groups. One of thereasons for that could be the presence of numerous par-alogous genes in many species of ascomycetes. Additionalefforts (i.e., broader sampling and inclusion of additionalspecies from basal lineages of Asomycota and Basidiomy-cota) will be required to produce a robust phylogenetic re-construction for this group.Below we will present and discuss the most important

findings of our analysis separately for each taxonomicgroup.

Phylum GlomeromycotaPhylum Glomeromycota is one of the oldest fungal lineagesthat is highly unusual in many ways. Members of this groupare obligate mutualistic symbionts forming a particular typeof mycorrhizal interaction, known as arbuscular mycorrhiza(AM) [16–18]. We have identified 34 genes and 19 pseudo-genes or gene fragments in the genome assembly ofRhizophagus irregularis, a species of Glomeromycotasequenced by the MGI consortium [18]. The distribu-tion of the identified genes among the recognised sub-families is shown in Table 2 and Additional file 2.Whereas the total number of ABC genes in Rh. irregularisis comparable with the numbers in other species includedin our analysis, we could not identify representatives ofsome of the groups that are commonly found in the ge-nomes of asco- and basidiomycetes (Additional file 1). Ab-sence of genes homologous to Saccharomyces cerevisiaeMDL1, YEF3 and YDR061W is particularly remarkable asthose groups are nearly ubiquitously present in the ge-nomes of asco- and basidiomycetes analysed so far [14, 15],and YEF3 (encoding the translation elongation factor 3)has shown to be essential for vegetative growth in S.cerevisiae [19]. We cannot exclude the possibility thatthe mentioned genes are present in the genome, but forsome reason they are missing from the available genomeassembly. Alternatively, some of them may have been lostby Rh. irregularis as a result of the adaptation to its obli-gate symbiotic lifestyle.Some aspects of the organisation of ABC genes in Rh.

irregularis show remarkable similarity with the zygomy-cete Rhizopus oryzae (Additional file 1) [14]. The simi-larity in the genome organization of Rh. irregularis andspecies of Mucoromycotina has been noticed previously

[18]. One of the most prominent common features ofRhizophagus and Rhizopus is a presence of four ABC-Dgenes. Nearly all asco- and basidiomycetes have onlytwo ABC-D genes. The products of those genes act as aheterodimeric peroxisomal long-chain fatty acid trans-porter. Remarkably, some of the species representingthe ancient evolutionary lineages within fungal king-dom (e.g., Rhizopus, Rhizophagus and chytridiomyceteSpizellomyces punctatus [14]) harbour additional genesof the subfamily ABC-D that in phylogenetic analysisare placed separately from the proteins of asco- and ba-sidiomycetes (Fig. 3). The origin and function of thoseadditional genes remains enigmatic.Little is known about the role of Rhizophagus ABC trans-

porters in the formation of arbuscular mycorrhiza. How-ever, the transcript profiling has shown that one of the Rh.irregularis ABC-B transporters is arbuscle-specific, i.e. it ishighly expressed in intraradical mycelium, but not duringother stages of the Rhizophagus life cycle [17].The unusual genetic organisation has substantially ham-

pered the genome assembly of Rh. irregularis [16], and theavailable assembly is highly fragmented [18]. Therefore,we cannot exclude that some of the identified genes repre-sent the different alleles of the same gene, and the actualnumber of ABC genes might be somewhat lower. At thesame time, the detected gene fragments might be ex-tended to full-length genes with further improvement ofthe genome assembly. It is also possible that some of thegenes are still missing from the assembly (in particular, wewould expect the presence of the ScYEF3 homologue inRh. irregularis genome).

Phylum AscomycotaClass Leotiomycetes/order HelotialesThree species from the order Helotiales were sequencedwithin the MGI project, Oidiodendron maius, Meliniomycesbicolor and M. variabilis. All three species form ericoidmycorrhiza with shrubs in the Ericaceae family (e.g.,Vaccinium spp. and Calluna vulgaris). Both Melinio-myces spp. can also colonise roots of forest trees (pine,spruce, birch etc.) forming ectomycorrhiza-like symbi-osis. The three species have the highest number of ABCgenes among the species included in our analysis (Table 2and Additional file 1). Interestingly, the number of ABCgenes in these species negatively correlates with the gen-ome size. O. maius has the highest number of ABC genes

Table 3 List of ABC-protein encoding genes significantly up-regulated in mycorrhiza-forming mycelium (MYC) as compared to free-living mycelium (FLM) (Continued)

603979 ABC-G PilcrABCG6.2 11.34 6.80E-07 1.2 13.3

Hebeloma cylindrosporum 19666 ABC-C HebcyABCC7.2b 5.61 1.09E-05 3.4 18.9

449030 ABC-D HebcyABCD1.1 3.22 0.020 6.2 20.0

448391 ABC-G HebcyABCG6.2 5.99 3.75E-04 9.9 59.4


(72 genes), but the smallest genome assembly size, whereasM. bicolor has the lowest number of ABC genes amongthe three species, but the largest genome (Additional file 1).

At the same time, M. bicolor has the highest number ofpseudogenes, and we have observed at least six cases whereM. bicolor ABC genes contained insertions of repetitive

Table 4 List of ABC-protein encoding genes significantly down-regulated in mycorrhiza-forming mycelium (MYC) as compared to free-living mycelium (FLM)

Species Protein model Subfamily Gene name Fold change p-value Mean FLM Mean MYC

Laccaria bicolor 633182 ABC-B (FL) LacbiABCB1.1a -2.68 0 10110 3774

617531 ABC-B (FL) LacbiABCB1.1b -9.59 6.43E-06 9869 1029

633953 ABC-B (HT) LacbiABCB4.1 -2.63 0 18225 6928

707098 ABC-B (HT) LacbiABCB5.1 -2.60 0 18954 7285

297457 ABC-C LacbiABCC2.1 -2.58 0 14297 5543

301178 ABC-C LacbiABCC3.1 -2.43 0.01 6912 2841

307663 ABC-C LacbiABCC3.2a -2.69 0 8401 3121

706294 ABC-C LacbiABCC3.2b -2.47 0.04 2312 937

305568 ABC-C LacbiABCC6.1 -2.21 0 11719 5311

Oidiodendron maius 193013 ABC-C OidmaABCC5.1d -2.23 5.17E-06 38.1 17.1

104920 ABC-G OidmaABCG1.1c -3.38 0.004 59.8 17.7

106738 ABC-G OidmaABCG1.1e -506.52 0 130.2 0.3

103241 ABC-G OidmaABCG3.1 -2.41 1.79E-05 30.3 12.6

Sebacina vermispora 68953 ABC-G SebveABCG7.2 -2.68 0 180.5 67.4

Tulasnella calospora 71373 ABC-A TulcaABCA1.1 -2.08 6.16E-04 28.7 13.8

22914 ABC-C TulcaABCC3.2a -2.14 0.014 15.5 7.2

32244 ABC-C TulcaABCC4.1 -2.20 3.21E-06 43.5 19.8

79164 ABC-D TulcaABCD1.1 -2.40 4.59E-11 69.2 28.9

234265 ABC-D TulcaABCD2.1 -2.88 4.63E-18 87.1 30.3

15356 ABC-F TulcaABCF3.1 -3.60 2.30E-11 131.0 36.3

Suillus luteus 628180 ABC-B (FL) SuiluABCB1.1 -2.43 3.02E-05 51.7 21.3

809813 ABC-B (HT) SuiluABCB4.1 -2.60 8.45E-25 239.6 92.1

799345 ABC-C SuiluABCC7.2 -2.23 1.23E-05 66.9 30.0

807343 ABC-G SuiluABCG2.1c -7.33 2.58E-60 219.9 30.0

807344 ABC-G SuiluABCG2.1d -2.22 3.29E-11 144.4 65.1

442329 Ydr061w SuiluABCI2.1 -4.14 3.95E-07 35.6 8.6

Piloderma croceum 820710 ABC-A PilcrABCA1.1a -2.08 9.23E-07 44.7 21.5

822387 ABC-B (FL) PilcrABCB1.1 -2.84 0 259.8 91.5

60709 ABC-C PilcrABCC3.2b -2.16 1.49E-10 67.0 31.0

801286 ABC-C PilcrABCC7.1 -3.80 0 141.2 37.1

816440 ABC-G PilcrABCG1.1 -7.40 2.12E-08 17.9 2.4

816614 ABC-G PilcrABCG2.1b -3.65 0 127.9 35.1

816747 ABC-G PilcrABCG3.1a -3.07 1.97E-09 36.4 11.9

813749 ABC-G PilcrABCG4.1 -4.20 0 102.4 24.4

Hebeloma cylindrosporum 441821 ABC-B (HT) HebcyABCB4.1 -2.00 0.039 33.9 17.0

440458 ABC-E HebcyABCE1.1 -2.58 1.49E-04 51.3 19.9

447601 ABC-F HebcyABCF1.1 -7.14 0.002 118.8 16.6

439681 ABC-F HebcyABCF2.1 -2.03 0.046 27.3 13.4

63066 ABC-G HebcyABCG4.1 -2.33 0.032 13.9 6.0

441280 ABC-I HebcyABCI1.1 -2.60 0.002 22.2 8.6


DNA (Fig. 6; Additional file 2). Extensive proliferation ofrepetitive DNA could be one of the factors contributing tothe increase of genome size in M. bicolor.A peculiar feature of all three species is a high number

of genes belonging to the group ABCC5 of ABC-C

transporters. Members of this group are often found asso-ciated with the secondary metabolism clusters. Indeed, wehave observed that 4 O. maius genes and one M. variabilisgene were located in the immediate vicinity of polyketidesynthase (PKS), nonribosomal peptide synthetase (NRPS)

CorglABCA1.1 HebcyABCA1.1a

HebcyABCA1.1b CC1G 00682

LacbiABCA1.1 LacamABCA1.1

TrimaABCA1.1AmamuABCA1.1

PilcrABCA1.1a ConpuABCA1.1a

SerlaABCA1.1 SuiluABCA1.1

PaxinABCA1.1 PaxruABCA1.1 SchcoABCA1.1 PilcrABCA1.1b

ConpuABCA1.1b HebcyABCA1.1c

PirinABCA1.1 SebveABCA1.1

TulcaABCA1.1 UM03454

OidmaABCA1.1c MelbiABCA1.1b

SNOG 01792 OidmaABCA1.1b

TubmeABCA1.1 ChoveABCA1.1

TerboABCA1.1 PTRG 02757 SNOG 03422

MelbiABCA1.1a MelvaABCA1.1b

CengeABCA1.1ANIG 08812

OidmaABCA1.1a MelvaABCA1.1a SS1G 12070

BC1G 13151 BC1G 11159

OidmaABCA1.2 MelbiABCA1.2

MelvaABCA1.2100100

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Fig. 1 Maximum-likelihood phylogenetic tree of ABC-A transporters. Numbers next to the branching points indicate the relative support from 100bootstrap replicates (only scores above 60 are shown). Ascomycetes- and basidiomycetes-specific branches are indicated. Selected fungal ordersare indicated by colour code. The fungal species are indicated with the following abbreviations: RO3G, Rhizopus oryzae; Gloin, Rhizomycesirregularis; ANIG, Aspergillus nidulans; SNOG, Phaeosphaeria (Stagonospora) nodorum; PTRG, Pyrenophora tritici-repentis; Cenge, Cenococcum geophilum;SS1G, Sclerotinia sclerotiorum; BC1G, Botryotinia fuckeliana (Botrytis cinerea); Melbi, Meliniomyces bicolor; Melva, M. variabilis; Oidma, Oidiodendron maius;Tubme, Tuber melanosporum; Chove, Choiromyces venosus; Terbo, Terfezia boudieri; CC1G, Coprinopsis cinerea; Amamu, Amanita muscaria; Corgl, Cortinariusglaucopus; Hebcy, Hebeloma cylindrosporum; Lacam, Laccaria amethystina; Lacbi, L. bicolor; Schco, Schizophyllum commune; Trima, Tricholoma matsutake;Serla, Serpula lacrymans; Conpu, Coniophora puteana; Boled, Boletus edulis; Paxin, Paxillus involutus; Paxru, P. rubicundus; Pismi, Pisolithus microcarpus; Pisti,P. tinctorius; Sclci, Scleroderma citrinum; Suilu, Suillus luteus; Pilcr, Piloderma croceum; Tulca, Tulasnella calospora; Pirin, Piriformospora indica; Sebve, Sebacinavermispora; UM, Ustilago maydis. Names of Saccharomyces cerevisiae genes are listed without additional indices. Filled diamonds next to the sequencenames indicate genes up-regulated in mycorrhiza-forming mycelium according to the transcriptomics data


or hybrid NRPS/PKS clusters (Fig. 7). At the same time,genes of this group were the ones most often targeted byinsertions of repetitive DNA, apparently resulting in theirpseudogenisation. We have detected this kind of insertionsin three M. bicolor genes and in one M. variabilis gene.The number of ABC genes in the three mycorrhiza-

forming species of the order Helotiales is considerablyhigher than in two phytopathogenic species from the sameorder, Sclerotinia sclerotiorum and Botryotinia fuckeliana(anamorph Botrytis cinerea) [14] (Additional file 1). Thehigher number of genes representing the group ABCC5 isprobably linked with the higher number of secondarymetabolism gene clusters in those species. Representa-tives of all four groups over-represented in mycorrhiza-forming Helotiales were identified among the geneswith the increased transcript abundance in mycorrhiza-forming mycelium (fold change > 2, FDR < 0.05) (Table 3;Additional file 6) [12]. This observation indicates that atleast some of them might have a role in the establishmentof the mycorrhiza symbiosis.

Class DothideomycetesCenococcum geophilum is the only known ectomycorrhi-zal species within the class Dothideomycetes. We havedetected 35 genes encoding predicted ABC protein to-gether with 5 putative pseudogenes or gene fragments inthe genome of C. geophilum (Table 2 and Additional file 1;Additional file 2). Contrary to the situation observed withinthe order Helotiales, the number of ABC genes in C.geophilum is somewhat lower than the correspondingnumbers in the genomes of the phytopathogenic dothi-deomycetes Pyrenophora tritici-repentis and Stagonosporanodorum [14]. Those differences are mainly due to thelower number of full-length ABC-B and ABC-G trans-porters in C. geophilum. Nevertheless, the sets of ABCgenes in the genomes of all three representatives of Dothi-deomycetes show high degree of similarity and there areonly a few noteworthy differences (Additional file 1).

Class Pezizomycetes/ order PezizalesThe order Pezizales is represented in our analysis bythree species, Tuber melanosporum (family Tubera-ceae), Choiromyces venosus (Tuberaceae) and Terfezia

PismiABCB2.1c PistiABCB2.1b

PistiABCB2.1a PistiABCB2.1c

PismiABCB2.1d PismiABCB2.1b

PismiABCB2.1a PistiABCB2.1d SclciABCB2.1a

SclciABCB2.1d SclciABCB2.1b

SclciABCB2.1c SclciABCB2.1e

BoledABCB2.1 PaxinABCB2.1b PaxruABCB2.1b

PaxinABCB2.1c PaxruABCB2.1a

PaxinABCB2.1a SuiluABCB2.1b

SerlaABCB2.1a ConpuABCB2.1a ConpuABCB2.1b

SuiluABCB2.1a SuiluABCB2.1c

PilcrABCB2.1e PilcrABCB2.1d

PilcrABCB2.1a PilcrABCB2.1b

PilcrABCB2.1c PilcrABCB2.1f

AmamuABCB2.1bAmamuABCB2.1c

AmamuABCB2.1eAmamuABCB2.1aAmamuABCB2.1d

CC1G 15513 CC1G 02511

CC1G 04855 CC1G 03025

TrimaABCB2.1 LacbiABCB2.1c LacamABCB2.1c LacbiABCB2.1b

LacamABCB2.1b CorglABCB2.1 HebcyABCB2.1

LacbiABCB2.1a LacamABCB2.1a

SchcoABCB2.1b SchcoABCB2.1a

SebveABCB2.1c SebveABCB2.1d

PirinABCB2.1 TulcaABCB2.1d

TulcaABCB2.1b TulcaABCB2.1a TulcaABCB2.1c SerlaABCB2.1b

UM06009 UM06461

GloinABCB2.1d GloinABCB2.1e

GloinABCB2.1f GloinABCB2.1a

GloinABCB2.1b GloinABCB2.1c

RO3G 06563 RO3G 05868 RO3G 09632

RO3G 15727 PTRG 02532 SNOG 05636

CengeABCB2.1c MelbiABCB2.1e

MelbiABCB2.1b CengeABCB2.1b

OidmaABCB2.1c MelbiABCB2.1cANIG 06436

CengeABCB2.1a MelbiABCB2.1d MelvaABCB2.1b OidmaABCB2.1b

MelvaABCB2.1cANIG 02300

TubmeABCB2.1 ChoveABCB2.1 TerboABCB2.1

OidmaABCB2.1d MelbiABCB2.1a

MelvaABCB2.1a OidmaABCB2.1a SS1G 07287 BC1G 15198

PTRG 12087 SNOG 15094

ANIG 02349ANIG 04091

MelvaABCB2.3e PTRG 05113 SNOG 06002

SS1G 13852 BC1G 05589

BC1G 10130ANIG 03608

TubmeABCB2.3a ChoveABCB2.3a

TerboABCB2.3 ChoveABCB2.3b

PTRG 10428 SNOG 05968

CengeABCB2.3 OidmaABCB2.3b MelvaABCB2.3b

ANIG 11826 SS1G 02000

BC1G 02708 MelvaABCB2.3c

MelbiABCB2.3a MelvaABCB2.3d

SS1G 04756 BC1G 00181 MelbiABCB2.3b

OidmaABCB2.3a MelbiABCB2.3c MelvaABCB2.3a

TubmeABCB2.2 ChoveABCB2.2 TerboABCB2.2

PTRG 08275 SNOG 02135 BC1G 03509

OidmaABCB2.2 MelbiABCB2.2

MelvaABCB2.2 UM05114

SS1G 06184 BC1G 10929

PTRG 01681 SNOG 14365

ANIG 00404ANIG 06237 SS1G 04252 BC1G 10568

UM05096 TulcaABCB2.4

GloinABCB1.1a GloinABCB1.1b

MelbiABCB1.1 OidmaABCB1.1

SS1G 04202 BC1G 00244 CengeABCB1.1

PTRG 07007 SNOG 00742

TerboABCB1.1 TubmeABCB1.1 ChoveABCB1.1

YKL209C UM04437

PirinABCB1.1a SebveABCB1.1

PirinABCB1.1b TulcaABCB1.1

SchcoABCB1.1a SchcoABCB1.1b

PaxinABCB1.1 PaxruABCB1.1

BoledABCB1.1 SclciABCB1.1 PismiABCB1.1 PistiABCB1.1

ConpuABCB1.1 SerlaABCB1.1 SuiluABCB1.1

CC1G 09456 PilcrABCB1.1

AmamuABCB1.1 TrimaABCB1.1 HebcyABCB1.1a CorglABCB1.1

HebcyABCB1.1b LacbiABCB1.1a LacamABCB1.1a

LacbiABCB1.1b LacamABCB1.1b

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Fig. 2 Maximum-likelihood phylogenetic tree of full-length ABC-Btransporters. Numbers next to the branching points indicate therelative support from 100 bootstrap replicates (only scores above60 are shown). The groups ABCB1 and ABCB2, the major clusterswithin them and ascomycetes-, basidiomycetes- and Rhizomyces/Rhizopus-specific branches are indicated. Selected fungal ordersare indicated by colour code. Please refer to the legend to theFig. 1 for the list of abbreviations of fungal names. Names of S.cerevisiae genes are listed without additional indices. Filled diamondsnext to the sequence names indicate genes up-regulated in mycorrhiza-forming mycelium according to the transcriptomics data


boudieri (Pezizaceae). All three species are characterisedby rather low number of ABC protein-encoding genes(Table 2 and Additional file 1; Additional file 2), and T.boudieri has the lowest number of ABC genes (29 genes)among mycorrhiza-forming species included in our ana-lysis and, as far as known, among all analysed filamentousascomycetes [14]. In particular, numbers of ABC-G, ABC-C and full-length ABC-B transporters in the analysed

species of Pezizales are lower than in other filamentousascomycetes (Additional file 1).The existence of 27 ABC transporters in the genome

of T. melanosporum has been reported previously [20].After careful examination and manual curation of theavailable gene models, we have identified five cases ofmisannotation, when a single ABC transporter-encodinggene was split into two separate non-overlapping models

PismiABCD2.1 PistiABCD2.1

SclciABCD2.1 BoledABCD2.1

PaxinABCD2.1 PaxruABCD2.1

SuiluABCD2.1 ConpuABCD2.1

SerlaABCD2.1 PilcrABCD2.1

AmamuABCD2.1 TrimaABCD2.1

CC1G 01702 HebcyABCD2.1

LacbiABCD2.1 LacamABCD2.1 SchcoABCD2.1

PirinABCD2.1 SebveABCD2.1

TulcaABCD2.1 UM01105

RO3G 01675 RO3G 04864

GloinABCD2.1 TerboABCD2.1

TubmeABCD2.1 ChoveABCD2.1

PTRG 05824 SNOG 07325

CengeABCD2.1ANIG 01014

OidmaABCD2.1 SS1G 10747 BC1G 04272

MelbiABCD2.1 MelvaABCD2.1

YKL188C MelbiABCD1.1 MelvaABCD1.1

OidmaABCD1.1 SS1G 05904 BC1G 12469

CengeABCD1.1 PTRG 04648 SNOG 11396

ANIG 10078 TubmeABCD1.1 ChoveABCD1.1

TerboABCD1.1YPL147W

RO3G 15916 GloinABCD1.1

UM03945 PirinABCD1.1

SebveABCD1.1 TulcaABCD1.1

PismiABCD1.1a PismiABCD1.1b

PistiABCD1.1 SclciABCD1.1 BoledABCD1.1

PaxinABCD1.1 PaxruABCD1.1

SuiluABCD1.1 ConpuABCD1.1

LacbiABCD1.1 LacamABCD1.1

CC1G 05451 SerlaABCD1.1

PilcrABCD1.1 TrimaABCD1.1

SchcoABCD1.1 HebcyABCD1.1

AmamuABCD1.1 RO3G 15328

GloinABCD3.1 GloinABCD4.1

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Fig. 3 Maximum-likelihood phylogenetic tree of ABC-D transporters. Numbers next to the branching points indicate the relative support from 100bootstrap replicates (only scores above 60 are shown). The groups ABCD1 and ABCD2 and ascomycetes-, basidiomycetes- and Rhizomyces/Rhizopus-specific branches are indicated. Selected fungal orders are indicated by colour code. Please refer to the legend to the Fig. 1 for thelist of abbreviations of fungal names. Names of S. cerevisiae genes are listed without additional indices. Filled diamonds next to the sequencenames indicate genes up-regulated in mycorrhiza-forming mycelium according to the transcriptomics data


(Additional file 2). After their correction, we concludedthat the genome of T. melanosporum has 24 genes forABC transporters and 8 genes encoding ABC proteinswithout transmembrane domains.The sets of ABC proteins in all three analysed species

of Pezizales show a high level of similarity, and in mostcases the corresponding homologues form well-supported groups in our phylogenetic analysis. The pres-ence of the genes representing the group ABCC4 in allthree species of Pezizales is noteworthy, as this type oftransporters is quite uncommon in ascomycetes.The transcript abundance of one of the T. melanosporum

ABC-G transporters was increased in the ectomycorrhizalroot tips compared to the free-living mycelium [20], indi-cating its potential role in the establishment of the mycor-rhizal symbiosis.

Phylum Basidiomycota/class AgaricomycetesOrder AgaricalesWe have analysed genome sequences of six mycorrhiza-forming species of Agaricales: Amanita muscaria, Corti-narius glaucopus, Hebeloma cylindrosporum, Laccariaamethystina, L. bicolor and Tricholoma matsutake. Thedata on L. bicolor ABC genes were taken from our previ-ous analysis [15]. All six species are engaged in ectomy-corrhiza symbiosis with a wide range of forest trees,including both conifers and broad-leaved trees. Thenumbers of identified ABC genes in the genomes ofmycorrhiza-forming Agaricales range from 33 genes inA. muscaria to 47 genes in C. glaucopus. All of the ana-lysed species, except for H. cylindrosporum, are charac-terised by the presence of high numbers of genefragments and putative pseudogenes related to the func-tional ABC protein-encoding genes, ranging from 15 inA. muscaria to 53 in L. amethystina (Additional file 1).There is a considerable similarity between the sets of

ABC genes in the genomes of the analysed mycorrhiza-forming species and of saprotrophic representatives ofAgaricales, Coprinopsis cinerea [14] and Agaricus bis-porus (our unpublished data), with the differences beingmostly quantitative. However, both saprotrophic specieshave a considerably lower number of putative pseudo-genes or gene fragments in their genomes. The set of

SuiluABCF1.1 ConpuABCF1.1

SerlaABCF1.1 PaxinABCF1.1

PaxruABCF1.1 BoledABCF1.1

SclciABCF1.1 PismiABCF1.1 PistiABCF1.1

AmamuABCF1.1 TrimaABCF1.1 PilcrABCF1.1 CorglABCF1.1 HebcyABCF1.1

LacbiABCF1.1 LacamABCF1.1

SchcoABCF1.1 CC1G 11006

PirinABCF1.1 SebveABCF1.1

TulcaABCF1.1 UM01341

RO3G 05654 GloinABCF1.1

YER036C TerboABCF1.1

TubmeABCF1.1 ChoveABCF1.1

ANIG 02210 PTRG 01254 SNOG 15381

CengeABCF1.1 SS1G 07508 BC1G 05115 OidmaABCF1.1 MelbiABCF1.1 MelvaABCF1.1

SS1G 04651 BC1G 00123 MelbiABCF2.1 MelvaABCF2.1 OidmaABCF2.1ANIG 04315

CengeABCF2.1 PTRG 00878

SNOG 07116 TerboABCF2.1 TubmeABCF2.1 ChoveABCF2.1

YFR009W RO3G 14239

GloinABCF2.1 UM03774 TulcaABCF2.1


CC1G 03722 CorglABCF2.1 HebcyABCF2.1

TrimaABCF2.1AmamuABCF2.1

LacbiABCF2.1 LacamABCF2.1

SchcoABCF2.1 PilcrABCF2.1

SerlaABCF2.1 PismiABCF2.1 PistiABCF2.1 SclciABCF2.1 ConpuABCF2.1

SuiluABCF2.1 BoledABCF2.1 PaxinABCF2.1

PaxruABCF2.1 GloinABCF3.1

PaxinABCF3.1 PaxruABCF3.1 SuiluABCF3.1

SerlaABCF3.1 ConpuABCF3.1

RO3G 05107 CC1G 15647

LacbiABCF3.1 LacamABCF3.1 HebcyABCF3.1

CorglABCF3.1a CorglABCF3.1b

SchcoABCF3.1 TulcaABCF3.1

UM02148 MelbiABCF3.1 MelvaABCF3.1 OidmaABCF3.1

SS1G 13151 BC1G 05922

ANIG 00948 TubmeABCF3.1

ChoveABCF3.1 PilcrABCF3.1

TrimaABCF3.1 CengeABCF3.1

PTRG 06739 SNOG 13131

PaxinABCF4.1 PaxruABCF4.1 BoledABCF4.1

SuiluABCF4.1 ConpuABCF4.1 SerlaABCF4.1 SchcoABCF4.1

PilcrABCF4.1 TrimaABCF4.1

HebcyABCF4.1 CorglABCF4.1 LacbiABCF4.1 LacamABCF4.1

CC1G 01993 PirinABCF4.1 SebveABCF4.1

TulcaABCF4.1 UM04253

RO3G 03347YPL226W

TubmeABCF4.1 ChoveABCF4.1 TerboABCF4.1

PTRG 05592 SNOG 07726

CengeABCF4.1ANIG 06651

SS1G 04907 BC1G 15506 OidmaABCF4.1

MelbiABCF4.1 MelvaABCF4.1

SS1G 13833 BC1G 15638

MelbiABCF5.1 MelvaABCF5.1

OidmaABCF5.1 CengeABCF5.1

PTRG 05025 SNOG 05861

TerboABCF5.1 TubmeABCF5.1 ChoveABCF5.1ANIG 06700

YLR249WYNL014W

RO3G 03289 RO3G 00750 RO3G 07209 RO3G 06471 RO3G 07483

UM04152 TulcaABCF5.1


CC1G 06090 SchcoABCF5.1 ConpuABCF5.1 PaxinABCF5.1 PaxruABCF5.1

BoledABCF5.1 PilcrABCF5.1

PismiABCF5.1 PistiABCF5.1 SclciABCF5.1

SerlaABCF5.1 SuiluABCF5.1

AmamuABCF5.1 TrimaABCF5.1 HebcyABCF5.1 CorglABCF5.1 LacbiABCF5.1 LacamABCF5.1

UM03351 MelbiABCE1.1 MelvaABCE1.1 OidmaABCE1.1 SS1G 06617 BC1G 04634

PTRG 00569 SNOG 06859

CengeABCE1.1ANIG 01256

TerboABCE1.1 TubmeABCE1.1 ChoveABCE1.1

YDR091C GloinABCE1.1 RO3G 12210 RO3G 13377

PirinABCE1.1 SebveABCE1.1

TulcaABCE1.1 SchcoABCE1.1 CC1G 07665 BoledABCE1.1 PaxruABCE1.1 PismiABCE1.1 PistiABCE1.1 SuiluABCE1.1 ConpuABCE1.1 SclciABCE1.1AmamuABCE1.1

PilcrABCE1.1 TrimaABCE1.1 SerlaABCE1.1 LacbiABCE1.1 LacamABCE1.1 CorglABCE1.1 HebcyABCE1.1

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Fig. 4 Maximum-likelihood phylogenetic tree of ABC-E and ABC-Fproteins. Numbers next to the branching points indicate the relativesupport from 100 bootstrap replicates (only scores above 60 are shown).The groups ABC-E, ABCF1, ABCF2, ABCF3, ABCF4 and ABCF5 andascomycetes-, basidiomycetes- and Rhizomyces / Rhizopus-specificbranches are indicated. Selected fungal orders are indicated bycolour code. Please refer to the legend to the Fig. 1 for the listof abbreviations of fungal names. Names of S. cerevisiae genes are listedwithout additional indices. Filled diamonds next to the sequence namesindicate genes up-regulated in mycorrhiza-forming mycelium accordingto the transcriptomics data


PaxinABCI2.1 PaxruABCI2.1 BoledABCI2.1

SerlaABCI2.1 SclciABCI2.1

ConpuABCI2.1 PismiABCI2.1 PistiABCI2.1

SuiluABCI2.1 PilcrABCI2.1

SchcoABCI2.1 TrimaABCI2.1

AmamuABCI2.1 CC1G 04547 CorglABCI2.1

HebcyABCI2.1 LacamABCI2.1

LacbiABCI2.1b LacbiABCI2.1d LacbiABCI2.1a

LacbiABCI2.1c TulcaABCI2.1

PirinABCI2.1 SebveABCI2.1 RO3G 11015

UM05951YDR061W

ChoveABCI2.1a ChoveABCI2.1b

TubmeABCI2.1 TerboABCI2.1

CengeABCI2.1 SNOG 14053 PTRG 08921

ANIG 00561 MelbiABCI2.1

MelvaABCI2.1 OidmaABCI2.1

SS1G 03677 BC1G 03532

RO3G 00630 GloinABCI3.1

TulcaABCI3.1 PirinABCI3.1

SebveABCI3.1 SchcoABCI3.1

PilcrABCI3.1 SerlaABCI3.1

LacbiABCI3.1 LacamABCI3.1

HebcyABCI3.1 CorglABCI3.1

CC1G 04237 TrimaABCI3.1 ConpuABCI3.1

SuiluABCI3.1 PismiABCI3.1

PistiABCI3.1 SclciABCI3.1 BoledABCI3.1 PaxinABCI3.1 PaxruABCI3.1

GloinABCI1.1 RO3G 00560

UM03153 PTRG 09757

SNOG 16144 CengeABCI1.1

ANIG 06512 SS1G 08753

BC1G 08185 OidmaABCI1.1 MelbiABCI1.1

MelvaABCI1.1 TubmeABCI1.1 ChoveABCI1.1

TerboABCI1.1YFL028C

AmamuABCI1.1 PirinABCI1.1

SebveABCI1.1 TulcaABCI1.1

SchcoABCI1.1 LacamABCI1.1

CorglABCI1.1 HebcyABCI1.1

CC1G 00284 TrimaABCI1.1

PilcrABCI1.1 SerlaABCI1.1

SuiluABCI1.1 ConpuABCI1.1

BoledABCI1.1 PaxinABCI1.1 PaxruABCI1.1

SclciABCI1.1 PismiABCI1.1

PistiABCI1.1

62

99

88

92

90

99

99

62

75

99

98

93

93

95

94

94

98

92

98

79

97

86

62

79

95

82

61

60

90

97

75

6574

75

98

60

74

0.5

Fig. 5 (See legend on next page.)


ABC genes in A. muscaria is quite distinct, as its gen-ome lacks some of the genes present in the remainingspecies (Additional file 1).The distinctive features of C. glaucopus genome are

the high number of the genes belonging to the groupsABCC4 and ABCC7, and the absence of ABC-D trans-porters and of the group ABCC6. The group ABCC4 ismade up by proteins of unknown function nearly ubiqui-tously present in Agaricomycotina. There are 11 genesof this group in the genome of C. glaucopus, whereastheir number in other analysed species of Agaricalesranges from 1 to 5. The group has undergone a remark-able amplification in Polyporales [15] with some specieshaving up to 16 genes.A common feature of the analysed species of Agaricales

(including saprotrophs) is the high number of genes be-longing to the group ABCB6 of half-size ABC-B trans-porters. To our current knowledge, the group is foundonly in the species of Agaricomycotina [15], and the bio-logical role of the corresponding proteins remains obscure.Analyses of the gene expression during mycorrhiza

formation have been performed in two species of

Agaricales, L. bicolor and H. cylindrosporum (Tables 3and 4; Additional file 6) [12]. The identification of a groupABCB6 half-size ABC-B transporter and ScYDR061W ho-mologues in L. bicolor experiment is noteworthy, as bio-logical functions of those two groups have not beenelucidated in any of the fungal species yet.

Order BoletalesWe have analysed the genomes of seven mycorrhiza-forming species of Boletales representing four distinctevolutionary lineages within the order [21]: Suillineae(Suillus luteus), Sclerodermatineae (Scleroderma citri-num, Pisolithus microcarpus and P. tinctorius), Paxilli-neae (Paxillus involutus and P. rubicundus), andBoletinae (Boletus edulis). We have also compared thedata from these 7 species with the sets of ABC trans-porters identified in two wood-degrading species ofBoletales (Coniophora puteana and Serpula lacrymans[15]) representing the basal lineages within the order[21]. The numbers of ABC-protein encoding genes inthe analysed mycorrhiza-forming species of Boletales rangefrom 30 in B. edulis to 42 in P. tinctorius (Table 2 and

(See figure on previous page.)Fig. 5 Maximum-likelihood phylogenetic tree of half-size ABC-I proteins. Numbers next to the branching points indicate the relative support from100 bootstrap replicates (only scores above 60 are shown). The groups ABCI1, ABCI2 and ABCI3 and ascomycetes- and basidiomycetes -specificbranches are indicated. Selected fungal orders are indicated by colour code. Please refer to the legend to the Fig. 1 for the list of abbreviations offungal names. Names of S. cerevisiae genes are listed without additional indices. Filled diamonds next to the sequence names indicategenes up-regulated in mycorrhiza-forming mycelium according to the transcriptomics data

Meliniomyces variabilis gene model 477483

1 5463

1 kbp

Meliniomyces bicolor gene model 641095

1 5178

Meliniomyces bicolor gene model 656557

1 7407

*

Fig. 6 Inactivation of an ABC transporter-encoding gene due to insertion of a transposable element. The scheme illustrates three closely relatedhomologous genes from the genomes of M. bicolor and M. variabilis. All three genes belong to the group ABCC5 of ABC-C transporters.The gene models Melva_477483 and Melbi_641095 are presumably intact, whereas the gene Melbi_656557 harbours a DNA transposoninsertion that likely has caused its inactivation. All three gene models share the same exon-intron structure with 16 exons shown as openrectangles. 5´- and 3´-untranslated regions in the model Melva_477483 are shadowed grey. The exon 3 in the model Melbi_656557 is disruptedby the insertion of repetitive DNA carrying a putative transposase-encoding ORF (shown by a filled arrow). In comparison with the models Melva_477483and Melbi_641095, the model Melbi_656557 also has three stop codons within the reading frame (indicated by filled triangles) and a putative translationalframe-shift (indicated by an asterisk). Note that the last exon in the model Melbi_656557 is truncated. Accumulation of these mutations probably occurredafter the gene has been inactivated by the transposon insertion


Additional file 1; Additional file 2). Genomes of many spe-cies (in particular, P. involutus and P. microcarpus) alsoharbour a high number of gene fragments and putativepseudogenes derived from ABC transporter-encodinggenes.There are no pronounced differences in sets of

ABC proteins between wood-degrading and mycorrhiza-forming species of Boletales. However, some of the ABCtransporters identified in Boletales have noteworthyfeatures. One of the interesting finding of our analysiswas the observation that the predicted proteins of thegroup ABCB4 of half-size ABC-B transporters in B.edulis, Paxillus spp., Pisolithus spp. and S. citrinum

differ considerably from their counterparts in other speciesof Agaricomycotina, including the remaining members ofBoletales (S. luteus, C. puteana and S. lacrymans). Se-quences from the six mentioned species were also clearlyseparated in the phylogenetic analysis, forming an isolatedbranch on the phylogenetic tree (Additional file 3). Re-markably, the sequences of their predicted transmembranedomains had a higher level of sequence similarity to theABCB4 proteins from other fungi than the sequences oftheir nucleotide-binding domains. Most plausible explan-ation for this observation would be that the changes in thenucleotide sequence ABCB4 gene occurred in the commonancestor of Boletineae, Paxillineae and Sclerodermatineae

Meliniomyces variabilis scaffold 36

7500 65000

ABC

5 kbp

NRPS

520727

642187

549728 615485 559381 234913

615523MFS

559383 503709

574030

Core cluster genes (NRPS/PKS)Other biosynthetic genesTransportersRegulatory genes

Oidiodendron maius scaffold 38

45000 114000

ABC NRPS

36363

139131

149988 173820 149992 139110 139128

PKS

Unknown function

62276

185052

46577

62279

139098

62281

46580 36373 46583


177000 227000

ABC PKS

149772

138176

184796

106945

71369

138097

173316 184800

173318 67262

46356

61792

173323

61797

61798


1175000 1230000

ABC PKS

31364

31365

181890 56940

56933

147060

181893

167366

31371

167368

128615 43476

43477

43492

105110

147066

128757MFS


470000 533000

ABCPKS-NRPS

99363

15956

150163

174030

46831

81615

46837

174035

46839

99369

MFS

139674

15958

21852

46835 99370

Fig. 7 Association of fungal ABCC5 transporters with the secondary metabolism gene clusters. The scheme shows four scaffold fragments of O.maius and a single fragment from M. variabilis. All five fragments are predicted to carry clusters of genes involved in the biosynthesis of secondarymetabolites (NRPS, PKS or hybrid PKS/NRPS clusters). The genes within the clusters are colour-coded according to their predicted function. All genemodel numbers are indicated. In all cases, genes encoding predicted ABC transporters are located in the immediate vicinity of the core gene ofthe cluster


after the separation of the lineage of Suillineae. It is not en-tirely clear what could be the cause of these changes, andwhether the derived genes retained their biological func-tion. The assignment of these proteins to the group ABCB4is tentative, and further analysis will be required to clarifytheir origin and phylogenetic affinities.Another distinct feature of Boletales is a presence of

the gene encoding an unusual ABC-C transporter with apredicted AMP-binding N-terminal domain. This genewas identified before in S. lacrymans, C. puteana andpolyporoid fungus Ceriporiopsis subvermispora [15]. Wecould detect this gene in all the analysed species of Bole-tales except for S. luteus. The identified sequences wereplaced in a separate isolated group in our phylogeneticanalysis. Remarkably, in all species the sequence codingfor the predicted AMP-binding domain is present, fur-ther strengthening our suggestion that this domain in-deed makes part of the transporter protein. It has beenhypothesised that this unusual structure could be essen-tial for tight coupling of the biosynthesis of yet unknownmetabolite with its transport, and indeed, the gene is as-sociated with an uncharacterised NRPS clusters in C.puteana and S. lacrymans [15]. However, we could notdetect similar associations of this gene with secondarymetabolism gene clusters in mycorrhiza-forming speciesof Boletales.Similarly to Agaricales, species of Boletales have a

diverse set of genes of unknown function belongingto the Agaricomycotina-specific group ABCB6 of half-size ABC-B transporters. Transcriptomics experimentshave identified several ABC genes of Boletales up-regulated in ectomycorrhiza-forming mycelium (Table 3;Additional file 6) [12].

Order AthelialesThe small order Atheliales is represented in our analysisby a single species, a broad host-range mycorrhizal sym-biont Piloderma croceum, widespread in boreal and tem-perate forests. Atheliales are most closely related toBoletales and Agaricales [22]. However, the number ofABC genes in Piloderma (48) exceeds the numbersfound in the analysed species of those two orders. It alsohas a considerable number (15) of gene fragments and pu-tative pseudogenes. The set of ABC transporters found inthe genome of P. croceum is characterised in particular bythe high diversity of ABC-G transporters, with some of thegenes without counterparts in other analysed species ofAgaricomycotina (Additional file 5). Other noteworthyfeatures of P. croceum are a high number of full-lengthABC-B transporters and a diverse set of half-sizeABCB6 genes. Three P. croceum ABC genes were sig-nificantly induced during mycorrhiza formation: full-length ABC-B (ABCB2) transporter, half-size ABC-B

transporter (ABCB6), and a half-size ABC-G trans-porter (ABCG6) (Table 3; Additional file 6) [12].

Order CantharellalesThis order is one of the ancient lineages within Agaricomy-cotina [22]. It is represented in our analysis by Tulasnellacalospora, a widespread symbiont of green orchids withthe world-wide distribution. In association with its hostplants, T. calospora forms a distinct type of endomycor-rhiza known as orchid-type mycorrhiza. The genome of T.calospora harbours 40 predicted ABC genes and 3 genefragments likely derived from the intact ABC genes. Theidentified set of ABC genes is in many aspects “ordinary”,i.e. there are no prominent distinctive features. All of thegroups of ABC proteins commonly found in Agaricomyco-tina are represented, and there are no “odd” genes. Wecould only mention here the relatively high number of full-length ABC-B genes, and the lower diversity of the groupABCB6 of half-size ABC-B transporters as compared withthe species of Agaricales and Boletales.Transcriptomics analysis has shown that 9 genes en-

coding ABC transporters and 2 genes encoding solubleABC proteins were up-regulated in T. calospora duringmycorrhiza formation (Table 3; Additional file 6) [12].

Order SebacinalesTogether with the order Cantharellales, this is one of thebasal lineages within Agaricomycotina. In our analysis, wehave included the orchid symbiont Sebacina vermifera,which was compared with the root endophyte Piriformos-pora indica [23]. The sets of ABC genes in the analysedspecies show considerable similarity (Additional file 1).The sequences of the half-size ABC-B transporters fromthe two species, which we provisionally assign to groupABCB4, are highly unusual and differ significantly fromthe corresponding sequences found in other Agaricomy-cotina, in particular in the amino acid sequence of theirnucleotide-binding domain. Both proteins are also con-siderably shorter than ABCB4 proteins in other speciesof fungi. In our phylogenetic analysis the two se-quences formed an isolated group of unclear relation-ship (Additional file 4). Their affinities with the groupABCB4 require additional studies.Transcriptomics analysis has identified a number of S.

vermifera ABC genes as being significantly up-regulatedin mycorrhiza-forming mycelium (Table 3).

DiscussionStudies on several model plant species have demonstratedthat particular plant ABC transporters are required for theformation of the arbuscular mycorrhiza [8–10]. At thesame time, the extensive exchange of nutrients betweenthe both partners engaged in mycorrhiza formation prob-ably requires the action of additional transporter proteins,


not only on the host side, but also on the side of the colo-nising fungus. Currently, it remains unknown whether anyof fungal ABC transporters contribute to the formation ofany type of mycorrhizal symbiosis.Our inventory provides an important insight into the di-

versity of ABC transporters and soluble ABC proteins inthe genomes of mycorrhiza-forming fungi. However, theinterpretation of the obtained results is considerably ham-pered by the scarcity of the available information on thebiological functions of fungal ABC transporters. Results ofour inventory indicate that there are no principal dif-ferences in sets of ABC proteins encoded by mycorrhiza-forming fungi, on one hand, and saprotrophic or phyto-pathogenic fungi, on other hand. We could not identify anyABC transporters that could be classified as “mycorrhiza-specific”. Moreover, the differences between members ofvarious evolutionary lineages of fungi are much more pro-nounced than between the species occupying different eco-logical niches. Thus, the principal question, whether any offungal ABC transporters play a role in the establishmentand functioning of mycorrhizal symbiosis, remains open.However, the available data on gene expression in sev-eral fungal species indicate that some ABC transporter-encoding genes are transcriptionally up-regulated duringthe mycorrhiza formation [12, 17, 20, 24]. The performedtranscriptomics studies have repeatedly identified mem-bers of some groups of ABC transporters as induced uponmycorrhiza formation, e.g. full-length ABC-B transportersof the group ABCB2, half-size ABC-B transporters of thegroup ABCB6, the transporters belonging to the groupABCG6 and the soluble ABC proteins of the groupABCI2. The group ABCB2 is widely distributed amongfungi and nearly ubiquitously present in members of dif-ferent lineages. Transporters of this group are also knownas MDR transporters, as some of them contribute to theresistance to antifungal compounds. The formation of par-alogous genes within this group is evident from our phylo-genetic analysis (Fig. 2). It is tempting to speculate thatsome of those paralogous copies have acquired new func-tions as a result of an adaptation to the symbiotic life style.The group ABCB6 is particularly interesting as it

seems to be specific to the species of Agaricomycotina.No members of this group have been identified up tillnow from other fungal lineages. Members of this grouphave a typical structure of half-size ABC transporters,with N-terminal transmembrane domain and C-terminalnucleotide binding domain. They show a considerabledegree of diversification within Agaricomycotina. In ourphylogenetic analysis, four clusters with high bootstrapsupport could be recognised within the group. The high-est diversity of its members is observed in the crowngroups of Agaricomycotina, i.e. orders of Agaricales andBoletales, whereas the diversity is lower in basal lineages(like Sebacinales and Cantharellales). ABCB6 genes have

been identified in all analysed Agaricomycotina regard-less of their life style and ecological niche with thesole exception of opportunistic pathogen Cryptococcusneoformans. None of the members of this group hasbeen characterised experimentally so far, but their widedistribution and a high diversity indicate that they mightplay an important role in basidiomycetes.ABCG6 is another group encompassing transporters

of unknown function. It is widely but unevenly distrib-uted among fungal lineages, as it is completely missing,for example, from all analysed species of Boletales. Thelast group ABCI2 is made up by the homologues of theuncharacterised S. cerevisiae gene YDR061W. Membersof this group are ubiquitously present in fungal ge-nomes, but their biological role remains obscure.Our analysis and the available transcriptomics data have

pinpointed those of ABC transporters that might contrib-ute to the mycorrhiza formation in different fungal line-ages. Whereas some of the transporters have beenrepeatedly identified in several species, there is no uniformpicture, and many of the genes have been up-regulated injust a single species. This is not particularly surprising, asmycorrhiza-forming fungi are very diverse phylogenetic-ally, and the ability to form mycorrhiza-like symbiosis hasapparently evolved independently in several evolutionarylineages of fungi [7]. Furthermore, there are considerablestructural and physiological differences between varioustypes of mycorrhiza (ectomycorrhiza, ericoid, orchid andarbuscular mycorrhiza), and fungi engaged in each of thosetype of symbiotic interactions likely have evolved their ownspecific adaptations. Additional work will be required toclarify the differences in nutrient flow and exchange and insignalling in various types of mycorrhiza symbiosis and, inparticular, contribution of different classes of transporterproteins to those processes.

ConclusionsWe report here the results of our large-scale effort to-wards the complete inventory of ABC protein-encodinggenes in the genomes of mycorrhiza-forming fungi. Re-sults of our inventory show that the sets of ABC genesdiffer considerably in various phylogenetic lineages, bothin total number of genes and in their distribution amongthe subfamilies. Genomes of Helotiales are particularlyrich in ABC transporter-encoding genes, having almosttwice as many corresponding genes as species of Pezizalesor some Boletales. Therefore, it is difficult to draw a com-mon pattern for all the analysed groups. The comparisonbetween the mycorrhiza-forming species and their sapro-trophic or parasitic relatives also revealed the considerabledifferences between various evolutionary lineages of fungi.Thus, mycorrhiza-forming species of Helotiales have highernumbers of ABC genes compared with the analysed plantpathogens of the same order, whereas in Dothideomycetes


we have observed an opposite trend (however, here thecompared species were not very closely related). In Agari-cales and Boletales the differences between the saprotrophsand mycorrhiza-formers were even less pronounced. How-ever, in many of the analysed species we have observed theformation of species- or lineage-specific groups of paralo-gous genes. Neofunctionalisation within such groups mightbe one of the forces contributing to the adaptation to thesymbiotic life style.Our results also contribute to the better knowledge of

the evolution and diversity of fungal soluble ABC proteinsand ABC transporters. We provide the first data on ABCprotein-coding genes for the members of one fungalphylum (Glomeromycota) and four fungal orders (Pezizales,Atheliales, Cantharellales and Sebacinales). The informationon ABC genes from the genome of the glomeromyceteRhizomyces irregularis is particularly important for theunderstanding of the evolution of this gene family inthe ancient lineages of the fungal evolutionary tree be-fore the divergence of lineages leading to modern Asco-mycota and Basidiomycota.Finally, the reported data should provide a contribution

to prioritising target genes for the forthcoming functionalcharacterisation, and in this way essentially advance thestudies of fungal ABC transporters in general.

MethodsGenome sequencingThe genomic DNA sequences of the mycorrhiza-formingfungi (except for the previously sequenced species Lac-caria bicolor [24], Tuber melanosporum [20] and Pirifor-mospora indica [23]) were obtained at the Joint GenomeInstitute (JGI) of the US Department of Energy (DOE) inWalnut Creek (California), as part of the Mycorrhizal Gen-omic Initiative (MGI) project. The genomes were pro-duced as described by [12]. The results from geneprediction and annotation in the above genomes are avail-able for searching in Mycocosm [25] at the JGI portal(http://jgi.doe.gov/fungi).

ABC genes identificationTo identify gene loci encoding ABC proteins in the fungalgenomes, multiple tblastn and blastp searches against se-lected genomes were performed at the website of the Fun-gal Genomics Program of the Department of Energy JointGenome Institute (JGI) [25]. Sequences of S. cerevisiaeand Coprinopsis cinerea ABC proteins representing allknown subfamilies were used as queries. All hits produ-cing E-values below 10-4 were further analysed. Genemodels were manually curated and, when necessary, thepositions of N and C termini and exon-intron boundarieswere adjusted.

Phylogenetic analysisPhylogenetic analysis was performed with the programpackage MEGA6 [26]. Initial assignment of identified se-quences to the subfamilies was done separately for everyspecies in a following way. All proteins identified in aparticular genome were aligned with known ABC pro-teins from S. cerevisiae, Aspergillus nidulans, C. cinereaand Ustilago maydis using MUSCLE algorithm inte-grated in MEGA6 package. Neighbor-joining phylogenetictrees were reconstituted based on the obtained alignments.All recognized subfamilies of ABC proteins could be sepa-rated on trees produced in such way, allowing the fastassignment of the analysed sequences to the particu-lar subfamily. The detailed analysis was performedseparately for each subfamily and, in the case ofABC-B transporters, also for full-length and half-sizeproteins. Multiple sequence alignments were constructedwith the MUSCLE algorithm integrated in MEGA6package using default settings. Alignments were qualitytrimmed with Gblocks 0.91 b [27]. Both full-length andtrimmed alignments were used to produce phylogenetictrees. Neighbor-joining trees were constructed usingJones-Taylor-Thornton substitution model with 500 boot-strap replications. Maximum-likelihood trees were ob-tained using Jones-Taylor-Thornton model [28] with 100bootstrap replications. Although the topologies of thephylogenetic trees produced with the different algorithmsshowed some minor differences, the same major groups ofABC proteins were recognized in all reconstructions.

Availability of supporting dataThe data sets supporting the results of this article are avail-able in the TreeBase repository under the following link:http://purl.org/phylo/treebase/phylows/study/TB2:S17878.

Additional files

Additional file 1: Table S1. Numbers of ABC protein-encoding genesin the analyzed species of fungi. (XLSX 16 kb)

Additional file 2: Table S2. Loci and amino acid sequences of ABCproteins identified in this work. (XLS 1484 kb)

Additional file 3: Figure S1. Maximum-likelihood phylogenetic tree ofhalf-size ABC-B transporters. Numbers next to the branching points indicatethe relative support from 100 bootstrap replicates (only scores above 60 areshown). The groups ABCB3, ABCB4, ABCB5 and ABCB6, the major clusterswithin them, the two outlying groups formed by the sequences fromBoletales and Sebacinales, and ascomycetes-, basidiomycetes- andRhizomyces / Rhizopus-specific branches are indicated. Selected fungalorders are indicated by colour code. Please refer to the legend tothe Fig. 1 for the list of abbreviations of fungal names. Names of S.cerevisiae genes are listed without additional indices. Filled diamondsnext to the sequence names indicate genes up-regulated in mycorrhiza-forming mycelium according to the transcriptomics data. (PDF 43 kb)

Additional file 4: Figure S2. Maximum-likelihood phylogenetic tree ofABC-C transporters. Numbers next to the branching points indicate therelative support from 100 bootstrap replicates (only scores above 60 areshown). The groups ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, ABCC6 and


http://jgi.doe.gov/fungi

http://purl.org/phylo/treebase/phylows/study/TB2:S17878

dx.doi.org/10.1186/s12862-015-0526-7

dx.doi.org/10.1186/s12862-015-0526-7

dx.doi.org/10.1186/s12862-015-0526-7

dx.doi.org/10.1186/s12862-015-0526-7

ABCC7, the outlying group ABCC7.5, and ascomycetes-, basidiomycetes-and Rhizomyces / Rhizopus-specific branches are indicated. Selected fungalorders are indicated by colour code. Please refer to the legend to the Fig. 1for the list of abbreviations of fungal names. Names of S. cerevisiae genes arelisted without additional indices. Filled diamonds next to the sequence namesindicate genes up-regulated in mycorrhiza-forming mycelium according tothe transcriptomics data. (PDF 1392 kb)

Additional file 5: Figure S3. Maximum-likelihood phylogenetic tree ofABC-G transporters. Numbers next to the branching points indicate therelative support from 100 bootstrap replicates (only scores above 60 areshown). Groups ABCG1-5, ABCG6 and ABCG7, the major clusters withinthem and ascomycetes-, basidiomycetes- and Rhizomyces / Rhizopus-specificbranches are indicated. Selected fungal orders are indicated by colour code.Please refer to the legend to the Fig. 1 for the list of abbreviations of fungalnames. Names of S. cerevisiae genes are listed without additional indices.Filled diamonds next to the sequence names indicate genes up-regulatedin mycorrhiza-forming mycelium according to the transcriptomics data.(PDF 1381 kb)

Additional file 6: Table S3. Expression levels of ABC protein-encodinggenes in free-living mycelium and in mycorrhiza-forming mycelium inthe selected mycorrhiza-forming fungi. (XLSX 56 kb)

Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionsAKovalchuk performed an identification and phylogenetic analysis of ABCproteins from the selected fungal genomes and drafted the manuscript.AKohler performed the analysis of expression data. FM and FOA conceivedof the study, participated in its design and coordination and helped to draftthe manuscript. All authors read and approved the final manuscript.

AcknowledgementsAcademy of Finland is gratefully acknowledged for research funding.The members of the Mycorrhizal Genomics Initiative consortium areacknowledged for making available their unpublished data on theexpression of ABC protein-encoding genes.

Author details1Department of Forest Sciences, University of Helsinki, P.O. Box 27, FIN-00014Helsinki, Finland. 2UMR 1136, INRA/Université de Lorraine, InteractionsArbres/Microorganismes, INRA, Institut National de la RechercheAgronomique, Centre INRA de Nancy, 54280 Champenoux, France.

Received: 13 April 2015 Accepted: 28 October 2015

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Diversity and evolution of ABC proteins in mycorrhiza ...

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