Research
Analysis of expressed sequence tags from the
ectomycorrhizal basidiomycetes Laccaria bicolor and
Pisolithus microcarpus
Blackwell Publishing Ltd.
Martina Peter1,4, Pierre-Emmanuel Courty1,4, Annegret Kohler1, Christine Delaruelle1, David Martin1,
Denis Tagu2, Pascale Frey-Klett1, Sébastien Duplessis1, Michel Chalot1, Gopi Podila3 and Francis Martin1
1
Unité Mixte de Recherche INRA–UHP 1136 ‘Interactions Arbres/Microorganismes’, Centre de Recherches de Nancy, 54280 Champenoux, France; 2INRA
Rennes, Unité Mixte de Recherche BiO3P, BP 35327, 35653 Le Rheu Cedex, France; 3Dept. of Biological Sciences, University of Alabama, Huntsville, AL
35899, USA; 4These authors contributed equally to this work
Summary
Author for correspondence:
Martina Peter
Tel: +33 383 39 40 80
Fax: +33 383 39 40 69
Email: peter@nancy.inra.fr
Received: 27 December 2002
Accepted: 8 April 2003
doi: 10.1046/j.0028-646x.2003.00796.x
• In an effort to discover genes that are expressed in the ectomycorrhizal basidiomycetes Laccaria bicolor and Pisolithus microcarpus, and in P. microcarpus/
Eucalyptus globulus ectomycorrhizas, we have sequenced 1519 and 1681
expressed sequence tags (ESTs) from L. bicolor and P. microcarpus cDNA libraries.
• Contig analysis resulted in 905 and 806 tentative consensus sequences (unique
transcripts) in L. bicolor and P. microcarpus, respectively. For 36% of the ESTs, significant similarities to sequences in databases were detected. The most abundant
transcripts showed no similarity to previously identified genes. Sequence redundancy analysis between different developmental stages indicated that several genes
were differentially expressed in free-living mycelium and symbiotic tissues of
P. microcarpus.
• Based on sequence similarity, 11% of L. bicolor unique transcripts were also
detected in P. microcarpus. Similarly, L. bicolor and P. microcarpus shared only a low
proportion of common transcripts with other basidiomycetous fungi, such as Pleurotus ostreatus and Agaricus bisporus. Such a low proportion of shared transcripts
between basidiomycetes suggests, on the one hand, that the variability of expressed
transcripts in different fungi and fungal tissues is considerably high. On the other
hand, it might reflect the low number of GenBank entries of basidiomycetous origin
and stresses the necessity of an additional sequencing effort.
• The present ESTs provide a valuable resource for future research on the development and functioning of ectomycorrhizas.
Key words: gene profiling, expressed sequence tags (ESTs), gene diversity, Laccaria
bicolor, Pisolithus microcarpus, ectomycorrhizal basidiomycetes.
© New Phytologist (2003) 159: 117–129
Introduction
Most tree species in temperate and boreal forests live in
symbiosis with ectomycorrhizal fungi. These fungi play a
crucial role in forest tree health by improving nutrient
acquisition, drought tolerance and pathogen resistance of
their plant hosts. In return, autotrophic plants provide their
heterotrophic fungal partners with carbohydrates (Smith &
Read, 1997). In the ectomycorrhizal association, fungal hyphae
© New Phytologist (2003) 159: 117–129 www.newphytologist.com
colonize absorbing fine roots of trees, develop a mantle around
these and penetrate between outer root cells forming
the so-called Hartig net. Extraradical hyphae spread into the
surrounding soil, take up nutrients and deliver them to the
ectomycorrhizal organ in which nutrient exchange takes place.
Many aspects of the ectomycorrhizal symbiosis have been
extensively studied both in ecological and physiological
respects (Smith & Read, 1997). The formation and functioning of the symbiosis include major changes in cellular and
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microcarpus (Coker & Mass.) Cunn. nom prov. isolate 441
(formerly P. tinctorius 441; Martin et al., 2002) were grown
and maintained on Pachlewski medium agar plates (Nehls &
Martin, 1995). For cDNA library construction of pure culture
mycelium, the isolates were transferred onto cellophanecovered agar plates containing high sugar (20 g l−1 glucose, 5 g l−1
maltose; L. bicolor) or low sugar (1 g l−1 glucose; P. microcarpus)
Pachlewski medium, and were grown for 3 weeks before
harvesting the proliferating hyphal tips at the periphery.
Ectomycorrhizas of P. microcarpus and E. globulus ssp. bicostata Kirkp. were synthesized using two previously described
in vitro systems (vertically or horizontally oriented agar plates;
Malajczuk et al., 1990; Burgess et al., 1996).
tissue morphology (Peterson & Bonfante, 1994), as well as in
the biochemistry and physiology of the partners (Botton &
Chalot, 1999; Hampp et al., 1999; Martin et al., 1999). Emerging genomic tools such as expressed sequence tags (ESTs) and
the cDNA array technology provide a new approach to the
understanding of ectomycorrhizal development and functioning at the molecular level (Martin, 2001). These techniques
allow to rapidly identify genes and to perform large-scale
functional analyses of thousands of them (Ewing et al.,
1999; Skinner et al., 2001). Because of the wide spectrum of
genes and signals involved, these genomic tools are well suited
to study molecular events in symbiotic interactions (Györgyey
et al., 2000; Voiblet et al., 2001; Podila et al., 2002).
At present, there is, however, a lack of a coordinated development of resources aimed at sequencing the genome of ectomycorrhizal fungi or producing genetic tools that are useful to
the scientific community (Martin, 2001). That is, the production of large numbers of ESTs or insertion and expression
tagged lines, as are available for several nonsymbiotic model
fungi. There is a relatively small (1642 ESTs published,
GenBank release 032103), albeit slowly accumulating, body
of EST sequence data derived from a number of ectomycorrhizal fungi in public (dbEST at the National Centre for Biotechnology Information [NCBI]) and in various local databases.
It includes ESTs for Hebeloma cylindrosporum (NCBI dbEST
& Sentenac et al. personal communication), Tuber borchii
(Lacourt et al., 2002; Polidori et al., 2002), Laccaria bicolor
(Podila et al., 2002), Amanita muscaria (U. Nehls et al., personal
communication), and Paxillus involutus ( Johansson et al.,
personal communication). To speed the discovery of novel
genes and functions involved in ectomycorrhiza development,
we have developed ESTs databases of 4-d-old Pisolithus
microcarpus /Eucalyptus globulus ectomycorrhizas (Tagu &
Martin, 1995; Voiblet et al., 2001).
In the project described here, we constructed cDNA libraries
and generated ESTs from the free-living mycelium of P. microcarpus
and of another ectomycorrhizal model species, L. bicolor. In addition, cDNA clones from P. microcarpus/E. globulus ectomycorrhizas at different developmental stages (4-, 12-, 21-d-old) were
partially sequenced to increase our current EST database of
Pisolithus ectomycorrhiza. Because the growth and mycorrhiza
development conditions were distinct, we expected to identify
a large number of novel genes. The gene diversity of the two
ectomycorrhizal basidiomycetes was assessed throught EST
sequencing, assembly and analysis. These data provide the basis
for an initial glimpse into the overall metabolism and biology
of fungal symbionts as revealed by expressed transcripts.
Total RNA was isolated from snap-frozen (liquid nitrogen) and
grounded fungal tissues using the RNeasy Plant Mini kit (Qiagen,
Valencia, CA, USA) or according to Logemann et al. (1987).
cDNA libraries of pure culture mycelium of L. bicolor and
P. microcarpus were constructed from total RNA using the
SMART cDNA synthesis kit in λTriplEx2 (Clontech, Palo Alto,
CA, USA). The resulting cDNA was packed into phages using
the Gigapack III Gold packaging kit (Stratagene, La Jolla, CA,
USA). Aliquots of the libraries were amplified, followed by
in vivo excision of the pTriplEx2 phagemid according to the
manufacturer’s instructions. Owing to the large proportion of
ESTs coding for rRNA genes in the library of L. bicolor
mycelium, a second cDNA library was constructed used poly(A)+
RNA. The mycelium was grown on low sugar Pachlewski medium
for 10 weeks, sampled and immediately frozen in liquid nitrogen.
Total RNA was extracted using the Qiagen Plant Mini kit and
poly(A)+ RNA was enriched from 13 µg of total Dnasedigested RNA, using the Qiagen Oligotex kit. cDNA library
was constructed in λTriplEx2 as previously described.
Several cDNA libraries were constructed of P. microcarpus/
E. globulus ectomycorrhizas. Three cDNA libraries were setup from total RNA of ectomycorrhizas formed in the horizontal Petri dish system (Malajczuk et al., 1990) and harvested
after 4 d of contact. These libraries and their construction
have already been described (Tagu et al., 1993; Voiblet et al.,
2001). Three additional libraries were constructed within the
framework of the present study, as described in the previous
section, from total RNA of ectomycorrhizas formed in the
vertical Petri-dish system (Burgess et al., 1996) and harvested
after 4-, 12-, and 21-d of contact between the symbionts.
Materials and Methods
DNA sequencing
Strains and culture conditions
The ectomycorrhizal basidiomycetes Laccaria bicolor (Maire
Orton) isolate S238N (Di Battista et al., 1996) and Pisolithus
Isolation of total RNA and construction of cDNA
libraries
Aliquots of the pTriplEx2 phagemid libraries were used for
infecting E. coli BM25,8 cells of OD600 1.0 and were
subsequently plated on Luria-Bertani (LB) agar containing
ampicillin. About 5000 bacterial clones from the various
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libraries were randomly collected, inoculated into 96-well
plates containing selective LB media, and grown overnight
without agitation at 37°C. Glycerol was added to a final concentration of 40%. Backup plates were created and stored at
−80°C. Aliquots of 3 µl were PCR (in 50 µl) amplified using
FORNAT (5′-AAGCGCGCCATTGTGTTGGTACCC-3′)
and REVALEX (5′-CGGCCGCATGCATAAGCTTGCTCG3′) as primers (present in the pTriplEx2 vector arms) (Kohler
et al., 2003). The PCR included 95°C for 3 min, 95°C for
60 s, 60°C for 30 s, 72°C for 3 min for 30 cycles and a final
extension at 72°C for 15 min (GeneAmp System 9700;
Perkin Elmer, Boston, MA, USA). Five µl of each reaction
were analysed on a 1% agarose gel and stained with ethidium
bromide to control the size and quality of the PCR products. Excess primers and nucleotides were removed by
ultrafiltration using the Montage 96-well plate system
(Millipore MAHV N45). Purified PCR products were
subjected to nucleotide sequencing on either a multicapillary
sequencer CEQ 2000XL (Beckman Coulter, Fullerton, CA,
USA) or on a ABI Prism 310 Genetic Analyser (Applied
Biosystems, Foster City, CA, USA). We used the FORNAT
primer, 50 bp upstream of the 5′ end of cDNA insert, and either
the CEQ Dye-labelled Dideoxy-Terminator Cycle Sequencing
kit ( Beckman Coulter), or the BigDye Terminator Cycle
sequencing kit and the POP-4 matrix (Applied Biosystems)
according to the manufacturer’s instructions.
The average length of readable sequences was 580 bp on
the CEQ 2000XL sequencer (P. microcarpus ESTs), and
390 bp on the ABI Prism 310 genetic analyser (L. bicolor
ESTs). Sequences obtained from mycelial cultures of
L. bicolor S238N were designated in the database with an ‘Lb’
at the beginning of the sequence identification number
(Lb01-Lb15, cDNA library constructed using total RNA;
Lb16-Lb30, cDNA library constructed using mRNA).
Sequences obtained from mycelial cultures of P. microcarpus
were designated in the database with a ‘P’, whereas ESTs
from 4-, 12-, and 21-d-old-ectomycorrhizas of P. microcarpus/
E. globulus were named ‘EP4’, ‘EP12’, and ‘EP21’, respectively. To this set of novel sequences, we added ESTs generated
in previously published studies (Nehls & Martin, 1995; Tagu
& Martin, 1995; Voiblet et al., 2001). ESTs from randomly
sampled cDNAs of 4-d-old P. microcarpus/E. globulus ectomycorrhizas, which were produced in the horizontal agar-plate
system (Tagu & Martin, 1995; Voiblet et al., 2001), were designated with an ‘St’, ‘un’, or no prefix. ESTs from cDNA
cloned using the suppressive subtractive hybridization (SSH)
procedure (Voiblet et al., 2001) or differential screening of
cDNA clones (Nehls & Martin, 1995) were designated with
an ‘EgPtd’ and ‘ud’, respectively.
Sequence processing and annotation
All sequence outputs obtained from the automated
sequencers were scanned visually to confirm overall quality of
© New Phytologist (2003) 159: 117–129 www.newphytologist.com
peak shape and correspondence with base calls. Sequence data
were uploaded in SEQUENCHER (version 3.1.1) (Gene
Codes Corporation, Ann Arbor, MI, USA) programme for
Macintosh. Leading and trailing vector and polylinker
sequences, and sequence ends with more than 3% ambiguous
base calls were removed by SEQUENCHER filters. ESTs
with less than 100 bp sequence information were eliminated.
Edited sequences were exported as FASTA text files for further
processing. The EST sequences were deposited in the
GenBank dbEST at the NCBI (GenBank accession no.
AW600807-AW600908, AW731605-AW731617, BE704426BE704449, BF707467-BF707495, BF942500-BF942695,
L41693-L41726, and CB009716-CB012283). A Mac OS
X-compatible programme, MacESTtools (available from
http://mycor.nancy.inra.fr/PoplarDB), was used for batch
execution of BLASTN and WU-BLASTX against the
nonredundant (nr) nucleic acid sequence databases at the
Baylor College of Medecine Web server (Worley et al., 1995).
This software was also used to retrieve best matches from the
output BLAST html files and generate datatables. Sequences
with an E-value < 1.0e−4 were considered to identify
known genes or have partial similarity to known genes.
Finally, MacESTtools was used to upload BLAST results in a
searchable MySQL database containing raw sequences and
BLAST results (http://mycor.nancy.inra.fr/EctomycorrhizaDB/).
The web site also provides the opportunity to search the
EST sequences using NCBI BLAST. The functions of ESTs
were assigned based on the BLASTX search and annotated
manually following the Munich Information Center for
Protein Sequences (MIPS) role categorization (http://
mips.gsf.de/proj/yeast/catalogues).
Assembly of the individual ESTs into groups of tentative
consensus sequences (TCs), representing unique transcripts,
was performed using the contig routine (80% identity over 40
nt length) of SEQUENCHER. The degree of amino acidsequence similarity between ESTs of various basidiomycetous
species was evaluated using the NCBI tBLASTX algorithm
in a stand-alone NCBI BLAST with executables for Apple
Macintosh downloaded from ftp://ftp.ncbi.nih.gov/blast/
executables/. The matrix BLOSUM62 and default settings of
BLASTALL was used to BLAST against locally created EST
databases of L. bicolor, P. microcarpus, and databases of ESTs
of Agaricus bisporus, Hebeloma cylindrosporum and Pleurotus
ostreatus retrieved from GenBank (release 121302).
Generation and analysis of cDNA arrays
Microarrays were produced separately for either L. bicolor or
P. microcarpus by spotting up to 1600 PCR-amplified cDNA
inserts onto nylon membranes using the BioGrid arrayer
(BioRobotics, Cambridge, UK) according to the manufacturer’s
instruction (Eurogentec, Saraing, Belgium). All unique
transcripts were spotted at least twice on membranes. For each
fungal species, total RNA was extracted from free-living
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mycelium grown on low sugar Pachlewski medium for
3 weeks. The transcript populations were amplified, labelled,
and hybridized to nylon microarrays as described (Lacourt
et al., 2002). Phosphorimages of hybridized membranes were
analysed in XDOTREADER (Cose, Paris, France) to obtain
background-subtracted raw spot intensity values.
Results and Discussion
cDNA library evaluation, cDNA sequencing and contig
analysis
Plasmid DNA from 4000 clones derived from mycelial
cDNA libraries of P. microcarpus 441 and L. bicolor S238N,
and from three cDNA libraries of 4-, 12-, and 21-d-old
P. microcarpus/E. globulus ectomycorrhizas was PCR amplified.
Up to 90% of the amplified clones contained a cDNA insert.
The inserts had an average size of c. 600 bp and 800 bp for
L. bicolor and P. microcarpus, respectively, with a size range
between 100 and 2400 bp. A total of 1519 L. bicolor clones
and 1681 clones of P. microcarpus and P. microcarpus/
E. globulus ectomycorrhizas were successfully sequenced
from the 5′ end. We removed contaminants corresponding
to mitochondrial and nuclear rRNA genes, as well as of
mitochondrial DNA (L. bicolor, 19%; P. microcarpus, 12%).
In addition, we have identified and eliminated E. globulus
sequences (193 ESTs, 19%) from the ectomycorrhiza databases based on macroarray analyses (Voiblet et al., 2001;
Courty et al., unpublished results), comparison to ESTs of
P. microcarpus mycelium, and BLASTN analysis against the
NCBI plant EST database. The remaining 1244 L. bicolor
and 1304 P. microcarpus sequences were further organized
in contigs to allow common clones to be identified. They
represented 905 and 806 tentative consensus sequences
(TCs), or unique transcripts, of L. bicolor and P. microcarpus,
respectively (Table 1). The number of ESTs in clusters ranged
between two and 75, with two and eight clusters including
more than 15 ESTs (> 1% of all transcripts) in L. bicolor and
P. microcarpus EST sets, respectively. Estimations suggest that
the genome of filamentous fungi, including ectomycorrhizal
fungi, averages 20 –40 Mb, with a complement of about
8000 genes (Kupfer et al., 1997; Le Quéré et al., 2002). Using
this approximation of the gene number in an ectomycorrhizal
fungus, the present TC sets corresponded to 10% of the total
expected complement of genes for L. bicolor and P. microcarpus.
The sequence redundancy (EST in clusters/total ESTs) reached
40% for both species and implies that continued sequencing
of random cDNA from our libraries still has the potential to
uncover novel sequences.
Functional characterization of expressed genes
To identify potential homologues to L. bicolor and
P. microcarpus genes, ESTs were compared to sequences
deposited in protein databases using the BLASTN and WUBLASTX algorithm (Worley et al., 1995). A total of 497 of
1244 L. bicolor ESTs (40%) corresponded to genes with
significant similarity to GenBank entries, including genes of
known function as well as hypothetical proteins of other
organisms. The remaining genes (60%) did not have
significant matches within the GenBank databases (Table 1).
Among the 1304 ESTs analysed of P. microcarpus, 647 (50%)
did not show any similarity to previously identified genes
within the NCBI database. The proportion of known genes in
the mycelial library was 34%, whereas it was higher in the
symbiotic tissues (40 –64%). Unknown genes represented
Table 1 The number of expressed sequence tags (ESTs) collected using different sampling strategies from Laccaria bicolor mycelium, Pisolithus
microcarpus mycelium, and Pisolithus microcarpus/Eucalyptus globulus ectomycorrhizas
ESTs
Sequence identity (WU-BLASTX/GenBank) of ESTs
Origin of ESTs
Total
TCsa
Clusters
Singletons
No
Low
match [1e−10 < x < 1e− 4]
Moderate
[1e−20 < x ≤ 1e−10]
High
[x ≤ 1e−20]
Laccaria mycelium, random
Pisolithus mycelium, random
Pisolithus/Eucalyptus
Mycorrhizas, totalb
4 d, random
4 d, SSH/DSc
12 d, random
21 d, random
Pisolithus totalb
1244
550
905
336
150
63
755
273
60%
66%
11%
9%
12%
8%
17%
17%
754
472
129
77
76
1304
539
347
56
71
66
806
67
39
18
3
6
147
472
308
38
68
60
659
37%
36%
24%
60%
39%
50%
10%
10%
15%
6%
6%
10%
11%
12%
8%
14%
12%
10%
42%
42%
53%
20%
43%
30%
a
TCs, tentative consensus sequences (= unique transcripts). bOnly EST originating from P. microcarpus. Discrimination among plant and fungal
origin of ESTs were based on macroarray hybridization intensities (Voiblet et al., 2001; P. E. Courty et al., unpublished), comparison with known
ESTs of P. microcarpus mycelium, and BLASTN analysis against the GenBank Plant EST database. cSSH, suppression subtractive hybridization;
DS, differential screening.
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50% to 65% of ESTs analysed in other filamentous fungi
(Ospina-Giraldo et al., 2000; Skinner et al., 2001; Lee et al.,
2002), whereas in plants, typically only 20 –25% of ESTs fall
into this category (Ronning et al., 2003; Kohler et al., 2003).
This might reflect the low number of GenBank entries of
fungal origin and emphasizes the value of the present
sequencing effort. The number of proteins with known
matches, found in both species and including all isoforms of
the same proteins (e.g. hydrophobins, metallothioneins), was
680. A complete list of genes identified with their BLAST
E-value is posted on the EctomycorrhizaDB Web site (http:/
/mycor.nancy.inra.fr/EctomycorrhizaDB/).
In silico transcriptional profiling
Digital analysis of gene expression can be performed by
counting the number of ESTs for a given gene within an EST
population from which transcript abundance can be inferred
(Ewing et al., 1999). This presumes that an important number (thousands) of ESTs is generated by randomly collecting
cDNA clones. Because EST data is inherently noisy, formulas
have been developed to estimate the statistical significance of
a detected differential gene expression between different EST
populations (Audic & Claverie, 1997). We performed digital
analysis of the gene expression by comparing randomly
sampled ESTs between the mycelia of the two ectomycorrhizal species L. bicolor and P. microcarpus, and between
mycelial and ectomycorrhizal ESTs of P. microcarpus. Although
the number of ESTs collected for these comparisons, ranging
between c. 600 and 1200, seemed to be enough to allow
digital profiling (cf. Audic & Claverie, 1997), the results may
change with further sequencing.
Transcripts expressed in P. microcarpus and L. bicolor
mycelium In the free-living mycelia of L. bicolor and P.
microcarpus grown on Pachlewski agar medium, the majority
of the most abundant transcripts, i.e. TCs with five or more
sampled ESTs, were novel genes for which no function could
be assigned (Table 2). Eight of the 15 most expressed genes of
L. bicolor, for example, were only found in this species so far.
The second most common transcript was already found in
L. bicolor isolate D170 and was assigned a ras-related protein
(Podila et al., 2002), but it showed low similarity to other
known ras GTPases from basidiomycetes (e.g. Suillus bovinus
GenBank no. AF250024; P. microcarpus, GenBank no. AF329890).
Six additional ESTs (TC_Lb01B20, TC_Lb05D07, histonelike protein, TC_Lb02B23, TC_Lb24H10, and TC_Lb25B08)
were expressed in both isolates S238N and D170 of L. bicolor
(Podila et al., 2002; G. Podila, unpublished data). Among
these 15 highly expressed transcripts, only one TC (CipC related
protein) was also detected in P. microcarpus. TC_Lb02B23
showed weak amino acid sequence similarity (E-value 1.0e−11)
to a Pleurotus ostreatus EST (GenBank n° AT002909; Lee et al.,
2002), whereas four transcripts (TC_Lb05D07, TC_Lb21F10,
© New Phytologist (2003) 159: 117–129 www.newphytologist.com
CipC related protein; and a ras-related protein) were similar
to Hebeloma cylindrosporum ESTs (GenBank no. BM077997,
BM078018, BU964309 and BM078038). Among prominent
transcripts with known function, two were involved in carbon
metabolism (cytochrome C oxidase; 1,4-benzoquinone reductase)
and one coded for a ribosomal protein.
In P. microcarpus mycelium, several of the most abundant
transcripts coded for structural proteins such as the hydrophobin HydPt-3 (GenBank no. AF097516) and the secreted
SnodProt1 protein. The latter displays a strong similarity
to the hydrophobin-related cerato-platanin, a phytotoxin
from the ascomycete Ceratocystis fimbriata (Pazzagli et al.,
1999). Other abundant TCs showed strong similarity to
metallothionein-related cysteine-rich proteins, which are likely
involved in metal transport, cellular detoxification and stress
response (Lanfranco et al., 2002). None of these transcripts
were detected in L. bicolor so far. This seemed to be the main
reason for the higher proportion of cell structure and cell/
organism defense proteins in P. microcarpus compared to
L. bicolor mycelium (5% vs 3%) when the proportion of ESTs
assigned to different functional categories were compared
(Fig. 1a). The proportion of transcripts coding for ribosomal
proteins and other components of the gene/protein expression machinery was strikingly different (7%, P. microcarpus;
13%, L. bicolor), which might indicate that the mycelium of
L. bicolor was more active at the sampling time.
Transcript abundance in the mycelium and symbiotic tissues of P. microcarpus Sequence redundancy analysis of
randomly sampled cDNA clones from the different developmental stages indicated that several genes were differentially
expressed in the free-living mycelium and symbiotic tissues of
P. microcarpus (Table 2; Fig. 1b). This is in agreement with
our previous studies on gene expression in P. microcarpus
mycelium and ectomycorrhizas using two-dimensional PAGE
(Hilbert et al., 1991), as well as suppression subtractive
hybridization (SSH) and macroarray analyses (Voiblet et al.,
2001). Significant differential expression was detected for two
(hydrophobin HydPt-2, SRAP17) of the five most abundant
transcripts found in ectomycorrhizas, which were not
detected in the free-living mycelium so far; both coded for
known symbiosis-regulated cell wall proteins (Martin et al.,
1999). By contrast, transcripts coding for a metallothioneinrelated cysteine-rich protein and SnodProt1 showed a significant lower expression in mycorrhizas compared to mycelium
(TC_11A2: mycorrhizas, 1.3%, mycelium, 5.5%; TC_P062A07:
mycorrhizas, 0.0%; mycelium, 1.5%). These results were
mirrored in the proportions of ESTs in the different
functional categories (Fig. 1b). They supported our previous
findings that the symbiotic interaction alters protein synthesis
(Hilbert et al., 1991; Voiblet et al., 2001) and the synthesis of
cell wall and extracellular matrix components in the fungal
partner (Laurent et al., 1999; Voiblet et al., 2001). The upregulation of the protein synthesis machinery and of central
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Table 2 Most abundant transcripts in Laccaria bicolor S238N free-living mycelium, in Pisolithus microcarpus 441 free-living mycelium, and in
Pisolithus microcarpus/Eucalyptus globulus ectomycorrhizas as determined by clustering of expressed sequence tags (ESTs)
EST abundance (%)
TCa #
Lb01B20
Lb02A14
Lb05D07
Lb26D11
Lb11E01
Lb24H10
Lb05E05
Lb02B23
Lb01F03
Lb10F12
Lb25B08
Lb21F10
Lb18H08
Lb21G12
Lb23D07
Lb05E10
Lb10G01
Lb17F10
Lb03F15
Lb18D02
Protein homologue (species)
L. bicolor mycelium
Hypothetical protein
Ras related protein (Laccaria bicolor)
Hypothetical protein
Arginine-rich, histone-like protein
(Parechinus angulosus)
Hypothetical protein, mucin-like
(Homo sapiens)
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
CipC related protein
(Emericella nidulans)
Hypothetical protein
40S ribosomal protein S12 (Blumeria graminis)
Proline-rich, LEA-like protein (Arabidopsis thaliana)
MAR-binding protein AHM1 (Triticum aestivum)
Cytochrome C oxidase subunit 1
(Agrocybe aegerita)
1,4-Benzoquinone reductase
(Phanerochaete chrysosporium)
Blast
E-value
L. bicolor
mycelium
P. microcarpus
mycelium
P. microcarpus
mycorrhizab
–c
1.3e−56
–
1.0e−06
1.9
1.3
1.1
1.0
0.0*
0.0*
0.0*
0.0*
0.0*
0.0*
0.0*
0.0*
1.1e−05
0.9
0.0
0.0
–
–
–
–
–
–
–
–
6.0e−14
0.8
0.8
0.6
0.6
0.5
0.5
0.5
0.5
0.5
0.0
0.0
0.0
0.0
0.0
0.0
0.2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.2
0.0
0.0
0.0
–
2.8e−24
9.4e−06
4.3e−08
1.4e−72
0.4
0.4
0.4
0.4
0.4
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
3.8e− 45
0.4
0.0
0.0
–
4.3e−05
0.0*
0.0*
8.7
5.5
4.6
1.3*
–
–
4.2e−23
2.7e−53
9.0e−48
0.0*
0.0*
0.0*
0.0*
0.0*
3.1
1.6
1.5
1.5
1.3
0.3*
1.0
0.0*
1.0
0.5
–
–
2.5e−20
–
–
0.0*
0.0*
0.1*
0.0*
0.0*
1.3
0.9
0.7
0.7
0.7
0.3
0.0*
0.8
0.0
0.3
P013H04
P063A06
P061B07
P063H10
P063G11
P014G02
P063G11
10C5
P. microcarpus mycelium
Hypothetical protein
Metallothionein-related protein
(Agaricus bisporus)
Hypothetical protein
Hypothetical protein
SnodProt1 (Neurospora crassa)
Hydrophobin HydPt-3 (Pisolithus tinctorius)
RNA-dependent RNA polymerase
(Helicobasidium mompa dsRNA mycovirus)d
Hypothetical protein
Hypothetical protein
Elongation factor 1-gamma (Artemia sp.)
Hypothetical protein
Hypothetical protein
(similar to hypothetical protein 7A3)
Cysteine-rich protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
–
–
–
–
–
–
–
–
0.0*
0.0*
0.0*
0.0*
0.0*
0.0*
0.0
0.0
0.7
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.2
0.3
0.3
0.0
0.0
0.2
0.0
0.2
ud240
5A8
11A2
5C4
P. microcarpus mycorrhizab
Hypothetical protein
Hydrophobin HydPt-2 (Pisolithus tinctorius)
Metallothionein-related protein (Agaricus bisporus)
Hydrophobin HydPt-3 (Pisolithus tinctorius)
–
6.4e−38
3.5e−05
2.1e−53
0.0*
0.0*
0.0*
0.0*
8.7
0.0*
5.5*
1.5
4.6
2.6
1.3
1.0
ud240
11A2
7A6
7A3
P062A07
5C4
EP1202B17
P012F05
P072E09
7A7
P013F06
P031A04
www.newphytologist.com © New Phytologist (2003) 159: 117–129
Research
Table 2 Continued
EST abundance (%)
TCa #
Protein homologue (species)
EgPtdB57
7A3
st54
SRAP 17
Hypothetical protein
Transmembrane FUN34 protein
(Schizosaccharomyces pombe)
Elongation factor 1-gamma (Artemia sp.)
60S ribosomal protein L10
(Saccharomyces cervisiae)
60S ribosomal protein L8 (Xenopus laevis)
WD-repeat GTPase CPC2 protein
(Neurospora crassa)
Ribosomal protein (Arabidopsis thaliana)
RNA-dependent RNA polymerase
(Helicobasidium mompa
ds RNA mycovirus)d
Hydrophobin HydPt-8 (Pisolithus tinctorius)
Hypothetical protein
60S ribosomal protein L24
Choline-P-cytidyltransferase (Brassica napus)
Protein kinase (Arabidopsis thaliana)
Hypothetical protein
Ubiquinol cytochrome C oxidoreductase
(Saccharomyces cervisiae)
7A7
8A9
5C2
6C8
5A1
EP1202B17
7C2
1E9
EP2102N0
6A1
9E6
7A6
7E3
Blast
E-value
L. bicolor
mycelium
P. microcarpus
mycelium
P. microcarpus
mycorrhizab
2.6e−50
–
1.1e−38
0.0*
0.0*
0.0
0.0*
1.6
0.4
1.0
1.0
0.8
2.5e−20
2.8e−83
0.1*
0.1
0.7
0.0
0.8
0.5
2.0e−98
2.8e−127
0.1
0.2*
0.0
0.2
0.5
0.5
6.5e−32
1.0e− 46
0.0*
0.0
0.0
1.3
0.5
0.5
6.4e−38
–
2.3e−72
5.2e−20
5.1e− 05
–
2.0e− 09
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.2
3.1
0.0
0.3
0.3
0.3
0.3
0.3
0.3
EST abundance = (ESTs in a cluster/total ESTs) × 100. Statistical significant (P < 0.05) differential expression based on the formula of Audic &
Claverie (1997) between corresponding tissues is indicated with an asterisk (EST abundance in bold face vs EST abundance marked with an
asterisk). Comparison of transcripts between L. bicolor and P. microcarpus were based on nucleotide- and amino acid-sequence similarity. aTC,
tentative consensus sequence; unique transcript bEST randomly collected from all mycorrhiza libraries, and only clones originating from
P. microcarpus. Discrimination among plant and fungal origin of ESTs was based on macroarray hybridization intensities (Voiblet et al., 2001; P.
E. Courty et al. unpublished), comparison with known ESTs of P. microcarpus mycelium, and BLASTN analysis against the GenBank Plant EST
database. cno significant similarity to GenBank entries (E-value = 1.0e−4). dTranscripts likely originating from double-stranded RNA mycovirus
(Osaki et al., 2002) expressed in P. microcarpus.
metabolic activities (Hampp et al., 1999, Fig. 1b) in mycorrhizas may explain why in the symbiotic fungal tissues, the
proportion of transcripts with GenBank homologues was
higher (mycorrhizas, 61%; mycelium, 34%).
An interesting unique transcript (TC_EP1202B17)
showed high similarity to a RNA-dependent RNA polymerase (Table 2). This transcript was identified as deriving from
a double-stranded RNA mycovirus expressed in the root rot
fungus Helicobasidium mompa (Osaki et al., 2002). The transcripts found in P. microcarpus are therefore likely to originate
from a mycovirus, which seemed to be highly expressed in
both mycelial and mycorrhizal tissues.
Digital transcript profiling vs microarray analyses To assess
whether the transcription level of genes, which were identified
as highly expressed in EST abundance analysis, could be
confirmed by microarray analysis, total RNA of free-living
mycelium of L. bicolor and P. microcarpus was extracted and
hybridized separately to cDNA arrays of the respective
species. We compared the 20 most highly expressed TCs
revealed by the two methods (Table 3). For L. bicolor, EST
© New Phytologist (2003) 159: 117–129 www.newphytologist.com
abundances in the two cDNA libraries constructed from freeliving mycelium under different growth conditions (library 1:
high sugar, grown for 3 weeks; library 2: low sugar, grown for
10 weeks) were separately analysed. The comparison revealed
that five of the 20 most abundant transcripts were detected as
highly expressed in all three mycelial tissues of L. bicolor,
irrespective of the analysis technique (Table 3a). Similarly,
eight TCs were identified by both methods to appear among
the 20 most abundant transcripts in P. microcarpus mycelium
(Table 3b). However, several transcripts identified as highly
expressed by microarray analysis were found in only one of the
two L. bicolor libraries among the 20 most abundant TCs. In
addition, some TCs were detected as highly expressed in only
one of the fungal mycelia studied, irrespective of the analysis
method used. Overall, these data indicated on the one hand
that in silico profiling was robust to detect the most abundant
transcripts (i.e. = 8 ESTs). On the other hand, they suggested
that results of digital profiling should be carefully interpreted,
since they are subjected to both, technical (e.g. due to limited
sampling of ESTs) as well as biological variation. Therefore,
unless verified by independent repetition using for example
123
124 Research
Fig. 1 Functional classification of expressed
sequence tags (ESTs) (a) from mycelial
cultures of Laccaria bicolor S238N and
Pisolithus microcarpus 441 and (b) from
free-living mycelium and symbiotic tissues of
Pisolithus microcarpus. For ectomycorrhizal
ESTs, randomly collected clones of various
mycorrhizal libraries (Tagu & Martin, 1995;
Voiblet et al., 2001) were included in the
analysis.
microarray analysis, RNA blot, or real-time PCR, the present
results should provide an initial glimpse into the gene
expression in the fungal tissues studied.
Analysis of shared transcripts between tissues, isolates
and species
Only a low percentage of shared transcripts (between 11%
and 26%; Table 4) was detected in free-living mycelium
grown under different conditions (e.g. L. bicolor grown on
high vs low sugar concentration) or in different tissues (e.g.
free-living mycelium vs symbiotic tissues) of the same fungal
isolate. Tissue-specific expression patterns, with 30 –90% of
all TCs to be differentially expressed, were reported in several
in silico and conventional gene expression studies of fungal
and plant species (Ewing et al., 1999; Ospina-Giraldo et al.,
2000; Lacourt et al., 2002; Lee et al., 2002). The reason for
such a low proportion of shared transcripts found in the
present study may be explained by the fact that only between
600 and 1000 ESTs of each tissue were sampled. This might
not be enough to represent the transcript populations in
the tissues, since the resulting 400–600 TCs corresponded to
only 5% of the total number of expected genes in basidiomycetes (Le Quéré et al., 2002). However, by sequencing
6000–20 000 ESTs of a single library (with EST redundancies of 80–95%), only a maximum 20% of the expected
number of genes from the respective plant (e.g. Solanum
tuberosum; Ronning et al., 2003) or fungal species (e.g.
Neurospora crassa; Zhu et al., 2001) was found in other
EST projects. In addition, whereas the number of detected
unique transcripts increased by sampling more ESTs of a library, the overall proportion of the most abundant transcripts
and functional categories were similar by sampling 1000 or
6000 ESTs of poplar roots (A. Kohler, personal communication). We therefore assume that augmenting the sequencing
effort will increase the number of shared transcripts between
tissues, but will still reveal differential gene expression between
them.
At the interspecies level, the percentage of shared TCs
between L. bicolor and P. microcarpus tissues amounted to 5%
based on nucleotide sequence similarity and to 11% based on
translated protein sequence similarity. The transcripts exhibiting the highest nucleotide sequence similarity between
these two species coded for genes involved in gene/protein
www.newphytologist.com © New Phytologist (2003) 159: 117–129
Table 3 Comparison of the 20 most abundant transcripts in Laccaria bicolor (a) and Pisolithus microcarpus (b) mycelium detected by expressed sequence tag (EST) abundance in libraries and
by cDNA microarray analysis
© New Phytologist (2003) 159: 117–129 www.newphytologist.com
(a)
L. bicolor
Mycelium library 1 (high sugar)
*
L. bicolor
Mycelium library 2 (low sugar)
**
L. bicolor
Mycelium microarray (low sugar)
I
*
Lb01B20
Lb05E05
Lb24H10
Lb02A14
Lb05D07
Lb10G01
Lb03F15
Lb01B11
Lb12C09
Lb11E01
Lb02B23
Lb23D07
Lb05E10
Lb03K04
Lb07A10
Lb29A12
Lb01L02
Lb03H14
Lb02A04
Lb12G05
Hypothetical protein
Hypothetical protein
Hypothetical protein
Ras related protein
Hypothetical protein
Proline-rich, LEA-like protein
Cytochrome C oxidase subunit 1
60S ribosomal protein
Hypothetical protein
Hypothetical protein, mucin-like
Hypothetical protein
Hypothetical protein
40S ribosomal protein S12
Glutathione S transferase 10
Transcription inititation factor TFA2
Glutathione S transferase
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
19/4
9/1
8/2
5/11
5/8
5/0
5/0
4/0
4/0
3/8
3/4
3/2
3/2
3/0
3/0
3/0
3/0
3/0
3/0
3/0
Lb02A14
Lb26D11
Lb05D07
Lb11E01
Lb25B08
Lb21F10
Lb21G12
Lb01F03
Lb18D02
Lb01B20
Lb02B23
Lb10F12
Lb18H08
Lb16B04
Lb19G03
Lb17F10
Lb05A07
Lb20A08
Lb19D11
Lb24H11
Ras related protein
Arginine-rich, histone-like protein
Hypothetical protein
Hypothetical protein, mucin-like
Hypothetical protein
Hypothetical protein
CipC related protein
Hypothetical protein
1,4-Benzoquinone reductase
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Profilins Ia/Ib
Elongation factor IA
MAR-binding protein AHM1
CipC related protein
Glycoprotein precursor
Ubiquitin fusion protein
60S ribosomal protein L41
11/5
10/2
8/5
8/3
6/0
6/0
6/0
5/2
5/0
4/19
4/3
4/2
4/2
4/0
4/0
3/2
3/2
3/1
3/0
3/0
Lb02A04
Lb01B20
Lb20A08
Lb03P16
Lb02B23
Lb12C09
Lb01O17
Lb05E05
Lb05D07
Lb26D11
Lb25B08
Lb02A14
Lb18E02
Lb11A02
Lb24H10
Lb02A07
Lb29A10
Lb02H20
Lb15C11
Lb01F03
Hypothetical protein
Hypothetical protein
Glycoprotein precursor
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Arginine-rich protein
Hypothetical protein
Ras related protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Polyphenol oxidase
Hypothetical protein
Hypothetical protein
Hypothetical protein
100
41
33
32
31
25
23
20
19
17
17
14
14
14
13
13
12
9
9
9
3/0
19/4
3/1
1/0
3/4
4/0
1/0
9/1
5/8
2/10
0/6
5/11
2/0
1/2
8/2
1/0
0/1
1/1
1/1
5/2
P. microcarpus
Mycelium library
***
P. microcarpus
Mycelium microarray
I
***
ud240
11A2
7A6
7A3
P062A07
5C4
EP1202B17
P012F05
P072E09
7A7
P013F06
P031A04
P013H04
P063A06
P061B07
P063H10
P063G11
P014G02
P063G11
10C5
Hypothetical protein
Metallothionein-related protein
Hypothetical protein
Hypothetical protein
SnodProt1
Hydrophobin HydPt-3
RNA-dependent RNA polymerasea
Hypothetical protein
Hypothetical protein
Elongation factor 1-gamma
Hypothetical protein
Hypothetical protein
Cysteine-rich protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
48
30
17
9
8
8
7
7
5
4
4
4
3
3
3
3
3
3
3
3
EP1202B17
EP1202P07
ud240
P061F11
EgPtdD43
5C4
P031D09
7A6
EP1201F05
8E5
EP402E10
11A2
P012D04
EP1202E05
EP1202P08
7A3
P013H04
P062A07
P012E09
st54
RNA-dependent RNA polymerasea
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein C24c9.13c
Hydrophobin HydPt-3
Hypothetical protein
Hypothetical protein
Farnesyl-pyrophosphate synthetase
Hypothetical protein
Hypothetical protein
Metallothionein-related protein
50S ribosomal protein
Hypothetical protein
EF-hand calcium binding peflin
Hypothetical protein
Cysteine-rich protein
SnodProt1
Beta GTPase
Transmembrane FUN34 protein
100
93
59
57
41
29
28
27
27
23
22
22
18
17
17
16
15
14
14
13
7
0
48
1
1
8
1
17
0
0
0
30
1
0
0
9
3
0
8
2
(b)
Research
125
Transcripts are sorted by descending order of their abundance. Gray scale indicates shared transcripts among the 20 most abundant tentative consensus sequences (TCs). * = Number of ESTs
in L. bicolor mycelium library 1/library 2; ** = Number of ESTs in L. bicolor mycelium library 2/library 1; *** = Number of ESTs in P. microcarpus mycelium library; I = absolute spot intensity
(highest value was set at 100). atranscripts likely originating from double-stranded RNA mycovirus (Osaki et al., 2002) expressed in P. microcarpus.
126 Research
Table 4 Comparison of tentative consensus sequences (TCs) between the same fungal tissue grown under different conditions, between
different fungal tissues of the same isolate, and between different fungal isolates
No. of TCs shared determined on
nt-sequence
levela
No. of TCs
No.
%b
Amino acid sequence level
(tBlastX; E-value ≤ 1e−10)
No.
%b
Laccaria bicolor S238N (mycelium, G20)
vs L. bicolor S238N (mycelium, G1)c
Pisolithus microcarpus (mycelium) vs P. microcarpus (mycorrhizas)
416 vs 428
61
15
69
17
336 vs 539
65
19
66
20
Pleurotus ostreatus (mycelium) vs P. ostreatus (fruitbody)d
650 vs 652
66
10
71
11
Agaricus bisporus (primordia) vs A. bisporus (fruitbody)e
245 vs 289
57
23
63
26
18
Laccaria bicolor S238N (mycelium) vs L. bicolor DR170 (mycelium)
f
905 vs 247
28
11
45
Laccaria bicolor S238N vs Pisolithus microcarpus
905 vs 806
37
5
87
11
Laccaria bicolor S238N vs Pleurotus ostreatus
905 vs 1256
74
8
126
14
Laccaria bicolor S238N vs Agaricus bisporus
905 vs 477
26
5
27
6
Laccaria bicolor S238N vs Hebeloma cylindrosporumg
905 vs 268
27
10
32
12
Pisolithus microcarpus vs Pleurotus ostreatus
806 vs 1256
54
7
129
16
Pisolithus microcarpus vs Hebeloma cylindrosporum
806 vs 268
21
8
29
11
Pisolithus microcarpus vs Agaricus bisporus
806 vs 477
28
6
35
7
a
Within the same fungal isolate: nucleotide-sequence identity ≥ 80%; between different fungal isolates: identity ≥ 60%. b(No of shared TCs)/
(smaller No of TCs of the two respective tissues) × 100. cG20, high sugar (20 g l−1 glucose, 5 g l−1 maltose) Pachlewski medium, mycelium grown
for 3 weeks; G1, low sugar (1 g l−1 glucose) Pachlewski medium, mycelium grown for 10 weeks dLee et al. (2002). eOspina-Giraldo et al. (2000).
f
ESTs of L. bicolor DR170: Podila et al. (2002) and G. Podila (unpublished data). gESTs derived from GenBank, release 121302, no corresponding
publication.
expression, such as ribosomal proteins (Table 5). Similar percentages of shared TCs were observed when ESTs of other
basidiomycetous fungi, retrieved from GenBank, were compared to the current sets of ESTs. At the protein sequence level,
6% (L. bicolor vs Agaricus bisporus) to 16% (P. microcarpus vs
Pleurotus ostreatus) of the transcripts were shared by two
species. As mentioned above, the high percentage of ESTs
without similarity to sequences of other fungal species might
result from the limited number of ESTs available so far. Furthermore, since the libraries were constructed from various
fungal tissues and from mycelium grown under different conditions, we assume that also the set of expressed transcripts
differed at least partially. To obtain a more accurate idea of
gene diversity in basidiomycetes, sequencing complete
genomes of basidiomycetous fungi would be necessary. Comparing 2555 TCs of Aspergillus fumigatus (30% of the total
number of predicted genes) to other ascomycetes whose
genomes were completely or almost completely sequenced,
such as Saccharomyces cerevisiae, Schizosaccharomyces pombe
and Candida albicans, Kessler et al. (2002) found only 30–
40% of the TCs to have a homologue in these fungi. By contrast, up to 70% of tomato and poplar ESTs showed a match
with Arabidopsis genes (Van der Hoeven et al., 2002; Kohler
et al., 2003). Tomato and Arabidopsis have diverged around
150 million years ago, which would be in the same range as
the divergence of Pisolithus and Laccaria (Bruns et al., 1998).
Whether the percentage of homologous genes between
L. bicolor and P. microcarpus is similar to the one observed
between plant species can only be answered with an additional
sequencing effort.
Conclusion
The present data provided a first global overview of the
gene diversity expressed in the mycelium of two model
ectomycorrhizal fungi, P. microcarpus and L. bicolor. EST
collections obtained from L. bicolor and P. microcarpus by
extraction of RNA from tissues exposed to different developmental and physiological conditions resulted in a considerable
variability in the most highly expressed transcripts. Therefore,
the analysis of ESTs not only of various ectomycorrhizal
species, but also of different tissues and tissues subjected to
different growth conditions is an efficient tool to discover
novel genes of these ecologically and economically important
fungi. The study confirmed that there is a lack of genomic
information of basidiomycetous, in particular ectomycorrhizal
fungi and revealed the necessity for an additional sequencing
effort. To our knowledge, there are several ongoing EST
www.newphytologist.com © New Phytologist (2003) 159: 117–129
Research
Table 5 Nucleotide sequence identity (≥ 60%) of 37 tentative consensus sequences (TCs) expressed in both basidiomycetous fungi Laccaria
bicolor and Pisolithus microcarpus
Functional categorya
Identity
(%)
nt-Seq
overlap
(bp)
Protein homolog (GenBank)
TC #
E
78
78
77
73
73
72
72
72
72
71
71
71
71
70
70
70
69
69
68
68
68
67
67
67
67
66
65
64
64
64
63
61
61
61
290
420
320
410
331
430
370
350
320
380
400
430
330
400
90
290
400
300
300
380
180
550
600
390
520
550
350
220
348
380
200
300
420
420
40S ribosomal protein S3ae-a
40S ribosomal protein S28
60S ribosomal protein L10
GTP-binding protein, beta subunit
40S ribosomal protein S11
60S ribosomal protein L26
60S ribosomal protein L27
60S ribosomal protein L20
Ubiquitin conjugating enzyme E2
Zinc metalloprotease
60S ribosomal protein L35
40S ribosomal protein S9-b
Glutamine synthetase
ATP/ADP carrier protein
60S ribosomal protein L2
60S ribosomal protein L18
Peptidyl-prolyl cis/trans isomerase
Glyceraldehyde 3-P dehydrogenase
Mitogen-activated protein kinase
60S ribosomal protein L44
Adenylate kinase b
Rho GDP-dissociation inhibitor 2
Arp 2/3 complex
Ubiquitin fusion protein
Transcription initiation factor IIa
Symbiosis-related protein
40S ribosomal protein S30
Elongation factor 1-gamma
Transcriptional regulator
Hypothetical protein
Small GTPase RAC1
Mitochondrial acyl carrier protein
CipC related protein
Ubiquitin-carboxy extension protein fusion
Lb03E23, EgPtdB53
Lb25A02, P064A05
Lb13H09, 7A9
Lb16E08, EgPtdA23
Lb18B07, 10C8
Lb23F05, 9A5
Lb21H04, P052D04
Lb03O03, EP1202K01
Lb02E18, EP2102F13
Lb27H04, EP402A10
Lb16G04, 7C3
Lb24C03, 9E3
Lb23E06, P011H04
Lb01B22, 10D4
Lb01L23, EP1202M06
Lb13G02, EgPtdD5
Lb11E03, P072G09
Lb22G04, 6D5
Lb16B01, P062C01
Lb08F01, EP1201H01
Lb19D05, P021A08
Lb01A06, EP402N08
Lb31D04, EP2102D05
Lb19D11, 12C3
Lb06D07, P062G03
Lb29B12, P031F04
Lb22E02, EgPtdB47
Lb15B12, P021F02
Lb01H19, EP2102H20
Lb16D04, P063F06
Lb20F12, P021D07
Lb02C21, EgPtdD19
Lb21G12, P011G10
Lb08C08, 3C5
x
x
x
M
C
S
F
NI
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
a
E, gene/protein expression; M, metabolism; C, communication/signaling; S, cell structure; F, cell fate; NI, no identification.
projects of ectomycorrhizal basidiomycetes (e.g. Paxillus
involutus, Hebeloma cylindrosporum), which will release large
sets of ESTs (3000–5000) within the next months (Tunlid
et al. and Sentenac et al. personal communications). Therefore,
we are positive that in the near future, more exhaustive
comparative analyses will help to complete the picture of the
gene diversity expressed in these fungal species. In the present
sets of ESTs, several abundant TCs, such as families of cell
wall components, hydrophobins and SRAPs, were already
characterized (Laurent et al., 1999; Martin et al., 1999), but
many yet unknown proteins await their functional delineation. The EST databases of L. bicolor and P. microcarpus contain many genes, such as transporters, assimilating enzymes
and transcriptional factors, which hold great promise to
elucidate the nutrient uptake and assimilation pathways in
© New Phytologist (2003) 159: 117–129 www.newphytologist.com
both, the free-living and symbiotic phases of these ectomycorrhizal fungi.
Acknowledgements
Martina Peter was supported by a postdoctoral fellowship
from the French Ministry of Foreign Affairs. Annegret Kohler
was supported by postdoctoral fellowships from the INRA
and the Région de Lorraine. The present investigation was
supported by grants from the INRA (Programmes ‘Sequencing
genomes of symbionts and pathogens’ and LIGNOME). The
research utilized in part the DNA Sequencing Facilities at
INRA-Nancy financed by the INRA, Région de Lorraine and
the European Commission. We would like to thank two unknown
referees for their constructive comments on the manuscript.
127
128 Research
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