Post on 10-Sep-2016
transcript
m y c o l o g i c a l r e s e a r c h 1 1 3 ( 2 0 0 9 ) 1 3 7 7 – 1 3 8 8
journa l homepage : www.e l sev i er . com/ loca te /mycres
Identification of heavy metal regulated genes from the rootassociated ascomycete Cadophora finlandica usinga genomic microarray
Markus GORFER*, Helene PERSAK, Harald BERGER1, Sabine BRYNDA2,Dragana BANDIAN, Joseph STRAUSS
Department of Applied Genetics and Cell Biology, Fungal Genomics Unit, Austrian Research Centers
and BOKU-University Vienna, Muthgasse 18, 1190 Vienna, Austria
a r t i c l e i n f o
Article history:
Received 6 July 2009
Received in revised form
4 September 2009
Accepted 15 September 2009
Available online 19 September 2009
Corresponding Editor:
Geoffrey Michael Gadd
Keywords:
Cadmium
Cadophora finlandica
Genomic microarray
Heavy metal
Lead
Zinc
* Corresponding author. Tel.: þ43 50550 36E-mail address: markus.gorfer@boku.ac.a
1 Present address: The Wellcome Trust, Ce2 Present address: Institut fur Molekulare
0953-7562/$ – see front matter ª 2009 The Bdoi:10.1016/j.mycres.2009.09.005
a b s t r a c t
The ascomycete Cadophora finlandica, which can form mycorrhizas with ectomycorrhizal
and ericoid hosts, is commonly found in heavy metal polluted soils. To understand the se-
lective advantage of this organism at contaminated sites heavy metal regulated genes from
C. finlandica were investigated. For gene identification a strategy based on a genomic micro-
array was chosen, which allows a rapid, genome-wide screening in genetically poorly char-
acterized organisms. In a preliminary screen eleven plasmids covering eight distinct
genomic regions and encoding a total of ten Cd-regulated genes were identified. Northern
analyses with RNA from C. finlandica grown in the presence of either Cd, Pb or Zn revealed
different transcription patterns in response to the heavy metals present in the growth me-
dium. The Cd-regulated genes are predicted to encode several extracellular proteins with
unknown functions, transporters, a centaurin-type regulator of intracellular membrane
trafficking, a GNAT-family acetyltransferase and a B-type cyclin.
ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction (Fauchon et al. 2002), Candida albicans (Riggle & Kumamoto
Soils are frequently contaminated by heavy metals (HMs),
mostly due to industrial activities (Anonymus 2005). Indepen-
dent of their origindeither from naturally metalliferous rock
or from anthropogenic pollutiondHMs in soil pose a challenge
to all kinds of soil organisms including bacteria, fungi, plants
and animals.
Most studies on how fungi deal with HMs were carried out
with model ascomycetes like Saccharomyces cerevisiae
27; fax: þ43 810 80 30 30.t
ntre for Cell Biology, Edin
Biotechnologie GmbH, Drritish Mycological Society
2000) and Schizosaccharomyces pombe (Bae & Chen 2004; Chen
et al. 2003; Coblenz & Wolf 1994; Ortiz et al. 1992) or with ecto-
mycorrhizal (ECM) basidiomycetes (Bellion et al. 2006). Only
few studies addressed HM gene regulation in soil inhabiting
ascomycetes like the ericoid mycorrhizal (ERM) fungus Oidio-
dendron maius (Vallino et al. 2005; Vallino et al. 2009).
Fungi have developed a complex defence system to coun-
teract cell toxicities by HM (for a recent review see Gadd
2007). The first barrier includes excreted substances like
burgh, UK.
.-Bohr-Gasse 3, 1030 Vienna, Austria.. Published by Elsevier Ltd. All rights reserved.
1378 M. Gorfer et al.
organic acids (Fomina et al. 2005) or probably proteins
(Gonzalez-Chavez et al. 2004) with an ability to immobilize
HMs. The second barrier includes (unspecific) binding of
HMs by the cell-wall (Bhanoori & Venkateswerlu 2000; Meharg
2003) and melanins located in the cell-wall (Fogarty & Tobin
1996). Toxic HMs that could not be detained outside the cell
must be detoxified inside the cell. These mechanisms include
complexation by glutathione (Fauchon et al. 2002), phytochela-
tins (Al-Lahham et al. 1999; Coblenz & Wolf 1994; Plocke & Kagi
1992) or metallothioneins (Bellion et al. 2007) and subsequent
disposal of the complex into the vacuole (Li et al. 1997; Ortiz
et al. 1992). The complexing agents are rich in cysteine amino
acids. In order to provide the cell with sufficient sulfur for the
synthesis of cysteines, sulfur is saved from other proteins by
downregulation of sulfur-rich proteins, a process described
in yeast as sulfur sparing (Fauchon et al. 2002). Additional to
detoxification and sequestration inside the cell, HMs can
also be exported (Riggle & Kumamoto 2000; Weissman et al.
2000). Notwithstanding this array of defence mechanisms,
HMs still can cause severe disturbances of essential cellular
functions, including inactivation of enzymes by binding to
sulfhydryl groups (Banerjee & Flores-Rozas 2005), displace-
ment of essential ligands in metallo-proteins (Vallee & Ulmer
1972) and generation of reactive oxygen species (Halliwell &
Gutteridge 1999). Therefore, fungal responses to HMs also in-
clude a cornucopia of repair mechanisms, like induction of su-
peroxide dismutase for removal of reactive oxygen species
(Jacob et al. 2001; Vallino et al. 2009) or activation of the protea-
somal protein degradation pathway for removal of damaged
proteins (Goller et al. 1998; Jungmann et al. 1993).
Complex regulatory networks have been identified, which
ensure that the metabolic costs of the HM response are kept
at a minimum without compromising the detoxification pro-
cess (Chen et al. 2003; Enjalbert et al. 2006; Gasch et al. 2000).
For a recent review on HM tolerance mechanisms of ECM
fungi see Bellion et al. (2006).
Cadophora finlandica (previously named Phialophora finland-
ica) (Harrington & McNew 2003) can form both ERM with eri-
coid hosts and ECM with ectomycorrhizal hosts (Vralstad
et al. 2002). It is frequently found in HM polluted habitats
and a possible functional role in HM resistance has been sug-
gested (Vralstad et al. 2002). The isolate used in this study
(C. finlandica PRF15) was obtained from an ECM root tip from
Salix caprea growing on a soil heavily contaminated with cad-
mium, lead and zinc (Dos Santos Utmazian et al. 2007). No se-
quence information except from the rRNA encoding cluster,
which is widely used as a phylogenetic marker, is currently
available from C. finlandica.
A genomic array approach was undertaken to identify HM
regulated genes from C. finlandica. The main advantages of ge-
nomic arrays are that (i) they can be used for identification of
regulated genes from genetically poorly characterized organ-
isms (arrays from not fully sequenced organisms based on
cDNA-libraries do not necessarily contain all ORFs!), (ii) gene
identification is possible even in mixed cultures in the pres-
ence of other organisms, (iii) regulatory elements of differen-
tially regulated genesdpromoters and terminatorsdcan be
obtained in one step and (iv) finally a genomic array can be
used for ChIP (chromatin immunoprecipitation) on chip ap-
proaches (Berger et al. 2008) bypassing the need for a highly
costly tiled genomic microarray. Since clones on a shot-gun
genomic array do not necessarily match transcriptional units,
shot-gun genomic arrays are primarily a tool for gene cloning
and not for transcriptomics. Expression levels of single genes
identified by genomic array hybridization must afterwards be
assessed by complementary methods like Northern analyses
or qPCR.
We describe here the construction of a C. finlandica shot-
gun genomic array and its use for the identification of HM reg-
ulated genes. In this work, growth under axenic conditions in
the presence or absence of Cd was chosen. A set of differen-
tially regulated microarray clones was then further analyzed
by sequencing and Northerns. Usually, more than one open
reading frame (ORF) was present in microarray clones with
an average insert size of 4.7 kb.
The availability of a gene transformation and tagging sys-
tem for C. finlandica (Gorfer et al. 2007) will allow the functional
characterization of selected HM regulated genes in the homol-
ogous host.
Materials and methods
Strains and culture conditions
The Cadophora finlandica strain PRF15, which was used
throughout this study, was previously characterized (Dos San-
tos Utmazian et al. 2007) and originates from an ectomycorrhi-
zal Salix caprea root tip growing on a soil heavily contaminated
with Cd, Pb and Zn in Arnoldstein (Austria). For RNA-isolation
C. finlandica PRF15 was pregrown on cellophane sheets on malt
extract agar (MEA, VWR) for two weeks at room temperature.
After this period, the mycelium was transferred with the cel-
lophane sheets to fresh MEA plates containing either of the
following metals in the indicated concentrations: 10 mM Cd,
100 mM Cd, 0.5 mM Pb, 5 mM Pb, 0.1 mM Zn or 1 mM Zn, con-
trol plates were MEA without the addition of HMs. The HMs
were added as nitrate salts. The higher HM concentrations
were chosen as concentrations under which unrestricted
growth still occurred without significant reduction, as judged
from colony diameter. After replacement, the plates were in-
cubated for another two days at room temperature before
RNA isolation.
Nucleic acids isolation
Genomic DNA from Cadophora finlandica PRF15 was isolated as
previously described (Gorfer et al. 2007). Further purification
was achieved by an RNase A (Qiagen) digestion followed by
phenol/chloroform/isoamyl alcohol and chloroform/isoamyl
alcohol extraction and ethanol precipitation. The pellet was
rinsed with 70 % ethanol and resuspended in 10 mM Tris pH
8.0. For RNA isolation, cellophane sheets with attached myce-
lial colonies were removed from agar plates and snap frozen
in liquid nitrogen and ground in liquid nitrogen to a fine pow-
der with a mortar and pestle. RNA isolation from the mycelial
powder was done with TRIzol (Invitrogen) according to the
manufacturer’s instructions. Integrity of RNAs was checked
on agarose gels.
C. finlandica genomic array 1379
Construction of the genomic microarray
Purified genomic DNA of Cadophora finlandica PRF15 was par-
tially digested with Mbo I (Fermentas) and protruding ends
were partially filled-in with dATP and dGTP with Klenow
DNA polymerase (Fermentas). The resulting fragments were
size-fractionated on an 0.8 % agarose gel and the desired frag-
ments in the size range between 4 and 6 kb were extracted
from the agarose gel with the Qiaex II gel extraction kit (Qia-
gen). The cloning vector pMS12 (Fungal Genetics Stock Center,
Kansas City, KS) was prepared by digestion with Xho I (Fer-
mentas) and partial fill-in with dCTP and dTTP Klenow DNA
polymerase (Fermentas). Vector and C. finlandica genomic
fragments were ligated and E. coli JM109 was transformed
with the ligation mixture. For the array a total of 10 848 colo-
nies from primary selection plates were picked and the plas-
mid DNA isolated by the alkaline lysis method (Birnboim &
Doly 1979). The inserts were amplified under the following
conditions: 1�DyNAzyme EXT Buffer detergent free (Finn-
zymes), 0.4 mM dNTP, 0.5 mM MS1201 (for primer details see
Supplemental Table S1), 0.5 mM M13fwd, 0.02 U/ml DyNAzyme
EXT DNA Polymerase (Finnzymes), 1.25 M betain with the
following PCR-program: initial denaturation at 95 �C for 203000
followed by 30 cycles of 94 �C for 4500, 56 �C for 10 and 72 �C
for 30 and a final extension at 72 �C for 100. All PCRs were
checked on agarose gels. 20 ml of PCR-products were dried
down, resuspended in 20 ml of spotting buffer (3� SSC; 1.5 M
betain) and spotted in duplicates onto Corning GAPS II slides.
Cy3-labelled oligonucleotides were applied as guide spots.
Positive controls included PCR-products for C. finlandica
PRF15 gpdA, tefA and actA. Microarray production was done
at the PICME, ARC GmbH.
General molecular genetic manipulations were carried out
according to Sambrook & Russell (2001).
Microarray hybridization and scanning
Cy3- and Cy5-labelled cDNAs were synthesized with the
CyScribe First-Strand cDNA-Labelling Kit (GE Healthcare)
and purified with the CyScribe GFX Purification Kit (GE Health-
care) according to the manufacturer’s instructions. Purified la-
belled cDNAs were lyophilized and resuspended in 10 ml
MilliQ-water. Slides were prehybridized in 5� SSC, 0.1 %
SDS, 1 % BSA at 45 �C for 1 h. Prehybridized slides were rinsed
five times in MilliQ-water, once in isopropanol and finally air-
dried. Competitive hybridization with Cy3- and Cy5-labelled
cDNAs from treatments with and without Cd in both combi-
nations (‘‘dye-swap’’) was carried out as follows: 10 ml of
each labelled cDNA was combined with 20 ml prewarmed
2�hybridization buffer (50 % formamide, 10� SSC, 0.2 %
SDS). The labelled cDNAs in hybridization buffer were heated
to 95 �C for 3 min and directly pipetted onto the microarrays.
Arrays were covered with cover slips and inserted into hybrid-
ization chambers. Hybridization was carried out at 42 �C over
night. The washing with the following buffers was done at
room temperature in a glass Coplin staining jar for the indi-
cated times: 2� SSC/1 % SDS (15 min), 1� SSC/0.2 % SDS
(8 min), 0.1� SSC/0.2 % SDS (5 min). After the final washing
step slides were dried by centrifugation in 50-ml-PP-tubes in
a swing-out rotor for 2 min at 450 g. The complete experiment
including the dye-swap was carried out twice. Arrays were
scanned using an Agilent G2565BA Microarray Scanner.
Microarray data analysis
Arrays were analyzed using Agilent Scan Control software
version 7.1.1 and the R software package version 2.1.1 (R De-
velopment Core Team 2005). Data from four microarray hy-
bridizations (dye swap plus technical replication) were first
normalized within arrays with the loess-function and then
normalized between arrays (Smyth & Speed 2003). Data for
replicate spots originating from the same genomic clone
were kept separate during data analysis. From the resulting
data a so-called topTable100 was created, which contained
a list of the 100 most strongly (up- or down-) regulated geno-
mic inserts. From this list a subset of genomic inserts where
both spots appeared in the topTable100 was selected for fur-
ther analyses.
RACE
For completion of incomplete ORFs on ends of the genomic in-
serts, mRNA from Cadophora finlandica grown in the absence
respectively presence of 100 mM Cd was amplified and end-
tagged with the SMART� mRNA Amplification Kit (Clontech/
TaKaRa) according to the manufacturer’s instructions. From
these cDNA-pools desired cDNA ends could be amplified
with a combination of gene-specific and tag-specific primers.
Northern analyses
For Northern analysis 10 mg total RNA was separated on
a formamide agarose gel, transferred to Zeta-Probe blotting
membranes (Bio-Rad) and fixed by baking at 60 �C for 2 h.
Probe DNA was generated by PCR amplification of the desired
genomic regions and radiolabelling with a32P-dCTP and
Ready-To-Go� DNA Labelling Beads (GE Healthcare). Hybrid-
ization was carried out as described by Church & Gilbert
(1984) at 65 �C. After hybridization membranes were washed
twice for 30 min in 1 mM EDTA, 40 mM Na2PO4 pH 7.2, 5 %
SDS and twice for 30 min in 1 mM EDTA, 40 mM Na2PO4 pH
7.2, 1 % SDS. After washing membranes were briefly dried on
Whatman 3 mm Chr filter paper, wrapped in cling film, ex-
posed to storage phosphor screens (GE Healthcare) and ana-
lyzed with the Storm phosphoimager (GE Healthcare). Signal
intensities were calculated with Quantity One 4.2 gel analysis
software (Bio-Rad).
Sequencing and sequence analysis
For sequencing of the genomic inserts from selected plasmids
transposons were inserted with the Template Generation Sys-
tem� II Kit (Finnzymes). Sequences starting from transposon
ends were generated with the BigDye� Terminator v3.1 Cycle
Sequencing Kit (ABI), separated on an ABI PRISM� 3100 Ge-
netic Analyzer (ABI) and assembled using VectorNTI software
(Invitrogen). Remaining gaps were filled by sequencing reac-
tions with specific primers. Signal peptides were predicted
with the SignalP3.0 server (www.cbs.dtu.dk/services/SignalP)
(Bendtsen et al. 2004), transmembrane domains with the
1380 M. Gorfer et al.
TMHMM server v. 2.0 (www.cbs.dtu.dk/services/TMHMM-2.0),
and secondary structures in nucleic acids with the DINAMelt
Server (dinamelt.bioinfo.rpi.edu/quikfold.php) (Markham &
Zuker 2005). Classification of transport proteins is based on
the Transporter Classification Database (www.tcdb.org) (Ren
et al. 2007). For primer design, Primer3 software was used (fok-
ker.wi.mit.edu/primer3/input.htm) (Rozen & Skaletsky 2000).
Results and discussion
Construction of a genomic microarray fromCadophora finlandica
To allow identification of regulated genes from C. finlandica
a strategy based on a genomic microarray was chosen. Since
no sequence information other than from the rRNA cluster
was available from C. finlandica, a set of putatively conserved
genes was PCR amplified and sequenced in order to obtain
an initial picture of genomic structure and organization. All
of the investigated genes, actA (actin, FJ860133), gpdA (glycer-
aldehyde-3-phosphate dehydrogenase GAPDH, FJ860132) and
tefA (translation elongation factor EF-1a, FJ860131) showed
an exon–intron–pattern and intron length highly similar to
orthologous genes from other Pezizomycotina. Likewise, codon
usage patterns for highly transcribed genes were similar to
patterns found in other Pezizomycotina (Edelmann & Staben
1994; Jekosch & Kuck 1999; Lloyd & Sharp 1991).
Based on this initial analysis the genomic DNA digestion
conditions using Mbo I restriction enzyme were chosen to ob-
tain a medium size for inserts in the genomic library of
w4.5 kb. It is known that medium gene densities from Pezizo-
mycotina range from 2.7 kb/gene for Fusarium graminearum to
4.0 kb/gene for Neurospora crassa (data from sequenced fungal
genomes at the Broad Institute, http://www.broad.mit.edu/
annotation/fgi/). Smaller inserts favour recovery of single
genes, which minimizes interferences of adjacent but differ-
entially regulated genes during hybridization and streamlines
the subsequent expression analysis. Bigger inserts on the
other hand favour recovery of complete genes including the
corresponding regulatory elements.
As a vector for the construction of the genomic library the
Aspergillus nidulans plasmid pMS12, which contains the auxo-
trophic argB-marker, was chosen. This strategy allows for
gene cloning by complementation in A. nidulans. Provided reg-
ulatory sequences from C. finlandica are recognized by A. nidu-
lans, heterologous expression can confer an increased
tolerance in A. nidulans. In this way, constitutively expressed
HM resistance determinants from C. finlandica that would oth-
erwise escape detection by the microarray cloning approach
can be identified.
An overview of the experimental steps starting from the li-
brary construction including quality control steps (see below)
to microarray hybridization, identification of differentially
regulated genomic regions and sequencing and transcrip-
tional profiling of the genomic regions is shown in Fig 1.
The actual average insert size of the plasmid-borne shot-
gun genomic library based on the analysis of 24 randomly
picked clones was 4.7 kb (see quality control QC1 in Fig 1).
No plasmids without inserts were found among the tested
clones. In a test library of w10 000 clones actA and tefA could
both be detected (QC2 in Fig 1). To obtain a good coverage of
the C. finlandica genome with an assumed size of 36 Mb (based
on data from sequenced Pezizomycotina genomes at the Broad
Institute, see above) a total of 10 848 single colonies were
picked and plasmid DNA was prepared. This resulted in a the-
oretical 1.4-fold coverage of the genome for the genomic
library. Due to a high failure rate of roughly 30 % during
PCR-amplification of the genomic inserts (QC3 in Fig 1), the
coverage on the prototype microarray slides dropped, how-
ever, to an estimated one-fold coverage of the genome. In fu-
ture series of the microarray this loss in genomic coverage can
probably be corrected by improving the PCR-conditions during
the amplification step.
Since genomic inserts on the array are random and borders
of the inserts do not necessarily correspond to transcriptional
units, output data after microarray hybridization cannot be
taken as transcriptional profiles. Instead, the genomic array
must be seen as a tool for the high throughput identification
of regulated genes. Subsequent analyses such as sequencing,
Northern analyses and/or Real-Time PCR must be included. A
similar approach was successfully undertaken for the identifi-
cation of regulated genes in Histoplasma capsulatum (Hwang
et al. 2003; Nittler et al. 2005) and can be regarded as an expe-
dient approach for organisms without completely sequenced
genomes.
Identification of Cd-regulated genomic regions bymicroarray hybridization
To identify genes regulated by exposure to HMs, pre-culti-
vated Cadophora finlandica colonies were exposed for two
days to 100 mM Cd on solid medium. These rather ‘‘mild’’ HM
exposure conditions, which do not significantly reduce
growth, and a comparatively long exposure period are differ-
ent to experiments carried out with yeasts (e.g. 1 mM Cd for
1 h in Saccharomyces cerevisiae (Fauchon et al. 2002), 0.5 mM
Cd for 10 min in Candida albicans (Enjalbert et al. 2006),
0.5 mM for up to 1 h in Schizosaccharomyces pombe (Chen et al.
2003)) but similar to the experimental conditions used for
the ectomycorrhizal basidiomycete Paxillus involutus (up to
44 mM Cd for up to 48 h (Jacob et al. 2004)). These conditions
seem appropriate when studying soil fungi. Bioavailable Cd
in soil is low and normally not strongly fluctuating. Soil fungi
at contaminated sites therefore encounter a long-term expo-
sure to comparably low, but nevertheless significant, amounts
of HMs.
Under these conditions, microarray hybridization compar-
ing cDNAs from exposed versus non-exposed mycelia resulted
in the identification of eleven Cd-regulated genomic inserts.
An exemplary plasmid insert is shown in Fig 2, whereas the
complete set of plasmid inserts is shown in Supplemental
Fig S1. Sequence analysis showed that two of the plasmids
(p083G03 and p085C08) contained identical inserts, and three
plasmids (p013A05, p042G08 and p105A02) contained overlap-
ping inserts. One plasmid-preparation (p001F09) consisted of
two independent plasmids, where only one of the two
(p001F09B) showed a HM-dependent regulation pattern. Data
from sequencing are summarized in Table 1. Additional
cDNAs were amplified from regulated genes located on
genomic DNA from C. finlandia PRF15
pMS12 partial digestion with MboI
digestion with Xho I partial fill-in with G and A
partial fill-in with C and T size-selection (4-6 kb) on agarose gel
QC1: determination of actualinsert size cloning
QC2: screening for representativegenes in test libraries
back-up of bacterial stocks at -80 °C
picking of single coloniesinto 113 MTP
back-up of plasmid DNA at -20 °C 113 x 96 plasmid preps
PCR-amplification of the inserts
QC3: evaluation of PCR on agarose gel
C.finlandia grown on MEA C. finlandia grown on MEA + 100 μM Cd
RNA isolation RNA isolation
labeling labeling
ligation
spotting of PCR-productsonto glass slides
genomic microarray
Northern analyses with fragments frominserts
sequencing of the complete insert
scanning
identification of reliable spots
plasmid isolation from bact. stockscorresponding to reliable spots
hybridization
data analysis
Fig 1 – Overview of experimental steps in the identification of Cd-regulated genes from Cadophora finlandica PRF15. QC1, QC2,
QC3: quality control steps during construction of the genomic microarray; MTP: microtiter plate; MEA, malt extract agar.
C. finlandica genomic array 1381
borders of the inserts to obtain the complete sequence infor-
mation. The overall gene density on the sequenced genomic
regions was found to be on average 1.8 genes per insert, and
this correlates well with the calculated density of 1.5 genes
per 4.5-kb-insert, a calculation based on gene densities from
sequenced Pezizomycotina genomes.
Transcription patterns of Cd-regulated genes fromCadophora finlandica
A comprehensive set of Northern analyses was performed with
sub-regions from Cd-regulated inserts. Fragments for Northern
hybridizations included both, regions with and without pre-
dicted open reading frames (ORF). Transcriptional profiling
was carried out with total RNA from mycelia grown in the ab-
sence of HMs and in the presence of either Cd (10 and 100 mM),
Pb (0.5 and 5 mM) or Zn (0.1 and 1 mM). These metals are present
atelevated levels in the Arnoldstein soil, from where C. finlandica
PRF15 was originally isolated (Wenzel & Jockwer 1999). Results
from Northern hybridizations are shown in Supplemental Fig
1 (complete set) and Fig 3 (only HM regulated genes).
From all genomic regions identified by microarray hybridiza-
tion as Cd regulated at least one Cd-regulated gene per insert
was found, although Cd-regulation of p025C09_ORF1 was very
weak: up-regulation to 135 % upon exposure to 10 mM Cd and
down-regulation to 89 % upon exposure to 100 mM Cd. Down-
regulation upon exposure to 100 mM Cd was expected from the
microarray hybridization results (M¼�0.86, see Table 1). Up-
regulation in response to Pb and Zn was found to be stronger
compared to Cd-dependent regulation (see Fig 3).
In two cases two independently regulated genes could be
found on one insert: p085C08 (and the identical clone
p083G03) and p075G11 both contain two genes, which are
down-regulated by Cd.
Fig 2 – Plasmid insert from p083G03 and p085C08. A map of the genomic insert in plasmids p083G03 and p085C08 is shown.
Open reading frames (ORFs) are indicated with exons and introns. Positions of primers as used for the amplification of
hybridization probes for Northern analyses are indicated by arrows ( and ) and the corresponding primer names. The
results of Northern analyses are shown below the map, with the conditions indicated on the left. Comparison was done to
the constitutively expressed genes for tefA and gpdA (Northerns shown on the right). The remaining plasmid inserts
including Northern hybridization results are shown in Supplemental Fig 1.
1382 M. Gorfer et al.
From the available expression data, five distinct classes of
HM regulated genes can be found in C. finlandica:
Class I: induced by Cd, but not by Pb or Zn
Class II: induced by Cd and Zn but not by Pb
Class III: repressed by Cd but not by Pb or Zn
Class IV: repressed by Cd, induced by Pb and Zn
Class V: repressed by Cd, Pb or Zn
For affiliation of genes to these classes see the following sec-
tion and Fig 3. No genes were found, which were up-regulated in
response to all three investigated metals. All three genes found
in Class V responded very strongly, with expression levels on Cd
dropping down to 8–39 % of the levels found in the absence of
HMs. Down-regulation was found independent of HM concen-
tration within the limits of this experiment. Analysis of more
genes will certainly increase the number of distinct classes.
Description of HM-regulated genes fromCadophora finlandica PRF15
Class Ip013A05_ORF1 (multidrug transporter): The hypothetical pro-
tein encoded by p013A05_ORF1 contains 12 predicted trans-
membrane (TM) domains and belongs to the drug:Hþ
antiporter family DHA1 (TC2.A.1.2.) within the major facilitator
superfamily (MFS) involved in drug efflux. The S. pombe CAF-5
protein, which shows highest similarity to p013A05_ORF1, is
a caffeine resistance protein from the DHA1-family, and the
gene also shows 3.6-fold induction upon exposure to Cd
(0.5 mM for 60 min, see Chen et al. 2003). Notably, not all S. pombe
drug transporter genes show this induction upon exposure to
Cd, some are down-regulated and some do not respond at all.
In S. cerevisiae Cd response is known to overlap with the oxida-
tive stress pathway, mediated by the Yap1 p transcription factor
(Hirata et al. 1994; Rodrigues-Pousada et al. 2004) and a set of
yeast multidrug transporters was shown to be regulated by
Yap1 p (Sa-Correia et al. 2009). Interestingly, the region 1 kb up-
stream of p013A05_ORF1 shows a DNA motif that perfectly
matches the characterized binding site for the yeast Yap1 p pro-
tein (TTASTAA; Fernandes et al. 1997) 327 bp upstream of the
start-ATG, making an overlap between Cd and oxidative stress
response also likely in C. finlandica. No putative metal responsive
elements (MRE: CTNTGCRCNCGGCCC; Stuart et al. 1985), or
stress responsive element (STRE: CCCCT; Martinez-Pastor et al.
1996) could be detected in the p013A05_ORF1 promoter region.
In the 30-UTR of p013A05_ORF1 an array of eight nearly perfectly
conserved direct repeats of 15 nucleotides is present (Fig 4). Sec-
ondarystructurepredictionscouldnot detect anystablesecond-
ary structures, into which the repeat region could fold. In the
p013A05_ORF1 mRNA the repeat region is therefore predicted
to form a single stranded conformation. No similar repeats
could be found in public DNA databases. The significance of
this repeat region is currently unknown.
p031E10_ORF1 (B-type cyclin): B-type cyclins regulate the
entry into mitosis (O’Connell et al. 1992). The 2.4-fold induc-
tion is probably a result of cell-cycle synchronization exerted
by Cd at the time point of the replacement from pregrowth in
Table 1 – Sequencing results for Cd-regulated regions from Cadophora finlandica.
Plasmid Acc. Nr. M� SD Fold Reg. ORFs Protein ID Length BLAST hit E value
p001F09A FJ860120 1.41� 0.111 2.66 1 reg. subunit proteasome rpn6
2 hypothetical protein
ACP19524
ACP19525
349-C
787-C
XP_001592823.1
XP_001592824.1
0.0
5e-103
p001F09B FJ860121 1 hypothetical protein
2 hypothetical protein (secreted)
3 phosphoribosylglycinamidsynthase
ACP19526
ACP19527
pseudogene
N-334
206
EED20761.1
XP_001912315.1
6e-81
2e-42
p013A05 FJ860122 a 1.51� 0.035 2.85 1 multidrug transporter ACP19528 618 XP_385494.1 0.0
p025C09 FJ860123 �0.86� 0.092 0.55 1 WSC-domain protein ACP19531 297 CAE76318.1 4e-96
p031E10 FJ860124 1.30� 0.004 2.46 1 B-type cyclin
2 Zn-finger protein (PHD)
ACP19532
ACP19533
352
276-C
XP_001550184.1
XP_001593122.1
7e-59
8e-42
p032E08 FJ860125 �1.02� 0.060 0.49 1 peptide transporter ACP19535 615 XP_001820585.1 0.0
p039H05 FJ860127 �0.87� 0.068 0.55 1 transposase
2 hypothetical protein (secreted)
pseudogene
ACP19536
233
120
EED17445.1
XP_001878943.1
8e-16
1e-27
p042G08 FJ860122 a 1.59� 0.242 3.01 1 multidrug transporter
2 Cupin 2 domain containing protein
ACP19528
ACP19529
618
172
XP_385494.1
XP_001816980.1
0.0
2e-25
p075G11 FJ860128 �2.86� 0.121 0.14 1 a-mannosidase
2 b-carbonic anhydrase (clade D)
3 centaurin b
ACP19537
ACP19538
ACP19539
N-389
171
1137
XP_001594341.1
XP_001911575.1
XP_001594268.1
0.0
5e-68
0.0
p083G03 FJ860129 b �0.99� 0.027 0.50 1 hypothetical protein (secreted)
2 phytanoyl-CoA dioxygenase
3 acetyltransferase (GNAT family)
ACP19540
ACP19541
ACP19542
294
312
234
XP_962131.1
XP_001593774.1
XP_001549514.1
9e-12
4e-117
1e-27
p085C08 FJ860129 b �0.88� 0.029 0.54 1 hypothetical protein (secreted)
2 phytanoyl-CoA oxidoreductase
3 acetyltransferase (GNAT family)
ACP19540
ACP19541
ACP19542
294
312
234
XP_962131.1
XP_001593774.1
XP_001549514.1
9e-12
4e-117
1e-27
p105A02 FJ860122 a 1.26� 0.077 2.39 1 cytochrome P450
2 multidrug transporter
ACP19530
ACP19528
30-C
618
XP_001400021.1
XP_385494.1
0.003
0.0
M� SD: M¼ log2(MEAþCd)� log2(MEA) or the log2 fold change in fluorescence intensity upon exposure of C. finlandica PRF15 to 100 mM Cd on MEA, where positive values indicate up-regulation and
negative values down-regulation. For convenience, M values converted to natural scale are shown in the row ‘‘Fold Reg.’’, where numbers>1 indicate up-regulation and numbers <1 down-regulation.
Values apply for complete genomic inserts (or even two independent inserts as it is the case for p001F09A and B).
Numbers given to ORFs correspond to numbers in Supplemental Fig S1. ORFs that were identified as Cd-regulated in subsequent Northern analyses are in bold text. Multidrug transporters from
p013A05, p042G08 and p105A02 are identical, inserts of these three plasmids contain overlapping regions of the genome. Plasmids p083G03 and p085C08 contain identical inserts.
Length: length of the conceptually translated protein in amino acids; incomplete protein sequences are marked by letters N resp. C depending to whether sequence corresponds to the N- or the
C-terminus.
BLAST hit and E value: accession number and E value of the highest scoring BLAST hit.
a The complete contiguous region covered by plasmids p013A05, p042G08 and p105A02 was submitted under accession number FJ860122.
b Plasmids p083G03 and p085C08 contain identical inserts, and were therefore only submitted once under accession number FJ860129.
C.
fin
lan
dica
gen
om
ica
rray
1383
gpdA
tefA
0 10 μ
M C
d
100
μM C
d
0,5
mM
Pb
5,0
mM
Pb
0,1
mM
Zn
1,0
mM
Zn
p001F09B_ORF2
hypothet. prot. (secreted)0
100200300400500600700800900
p013A05_ORF1
multidrugtransporter0
100
200
300
400
p025C09_ORF1
WSC domain protein 0
100
200
300
p031E10_ORF1
B-type cyclin 0
100
200
300
p032E08_ORF1
peptide transporter0
100
200
p039H05_ORF2
hypothet. prot. (secreted)0
100200300400500
p075G11_ORF3
centaurin
p075G11_ORF2
-carbonic anhydydrase0
50
100
150
p085C08_ORF3
acetyl-transferase 0
50
100
150
p085C08_ORF1
hypothet. prot. (secreted) 0
50
100
150
200
Class I
Cd ↑ Pb ↔ Zn ↔
Class II
Cd ↑ Pb ↔ Zn ↑
Class III
Cd ↓ Pb ↔ Zn ↔
Class IV
Cd ↓ Pb ↑ Zn ↑
Class V
Cd ↓ Pb ↓ Zn ↓
0
50
100
150
β
β
1384 M. Gorfer et al.
Fig 4 – Repeat region in the 30-UTR from p013A05_ORF1. The
stop codon is underlined, the repeat region is highlighted by
bigger letter font.
C. finlandica genomic array 1385
the absence of Cd to a growth-medium containing Cd. Cell-
cycle synchronization is either a Cd-specific effect, or a general
effect of the replacement procedure, but with differences in
cycle-frequencies after release of the arrest depending on
the type of HM. Cd-exposed mycelium was than sampled at
a cell-cycle stage high in p031E10_ORF1 mRNA, whereas the
other mycelia were sampled at a cell cycle stage low in
p031E10_ORF1 mRNA.
Class IIp001F09B_ORF2 (hypothetical protein, secreted): p001F09B_ORF2
belongs to a family of conserved fungal proteins with a size of
w220 aa and no known function. Proteins are predicted to con-
tain a cleavable signal peptide. The p001F09B_ORF2 related pro-
tein from Coccidioides immitis, ELI-Ag1, was identified as
a protective antigen after expression library immunization in
mice (Ivey et al. 2003) further corroborating the exposure of these
proteins to the cell-surface, as already suggested by the predic-
tion of a cleavable signal-peptide. The ELI-Ag1-like protein from
Histoplasma capsulatum shows down-regulation upon exposure
to Cu (Gebhart et al. 2006). While the response of p001F09B_ORF2
to Pb was rather weak, up-regulation upon exposure to Cd was
strong (3.6-fold at 100 mM Cd) and even stronger upon exposure
to Zn (8.8-fold at 1 mM Zn). Cu was not tested in our experiments.
Class IIIp085C08_ORF1 (hypothetical protein, secreted): p085C08_ORF1
is predicted to be an extracellular protein with a cleavable sig-
nal peptide. Potential N-glycosylation sites are not found. The
central core (aa 79–263) is enriched in threonine. Most of the
Fig 3 – Northern analysis of heavy metal regulated genes from C
the figure. For the complete set of Northerns refer to Suppleme
defined in the text, depending on differential expression in res
ative expression levels with mRNA abundance of the no-heavy
(shown on the top) were used for normalization.
homology to published protein sequences is restricted to this
central threonine-rich core.
Class IVp025C09_ORF1 (WSC-domain proteins): p025C09_ORF1 is pre-
dicted to be an extracellular protein with a cleavable signal
peptide and contains three WSC domains, which are involved
in carbohydrate binding, cell wall integrity and stress re-
sponse. WSC domains are often combined with other domains
(e.g. copper radical oxidase in Phanerochaete chrysosporium
(Vanden Wymelenberg et al. 2006) or an exo-b-1,3-glucanase
in Trichoderma asperellum (Bara et al. 2003)), but WSC-domain-
only proteins do exist e.g. in Chaetomium globosum (c.f.
XP_001221976, 4 WSC-domains). Cd-dependent regulation
was found to be very weak, with a slight up-regulation at
10 mM Cd and a slight down-regulation at 100 mM Cd. Although
the down-regulation found in the Northern experiments is
probably not significant, it is consistent with the data obtained
from array hybridization. Regulation by the other tested
metals, Pb and Zn, was found to be stronger than the effect
exerted by Cd. The lower concentrations (0.5 mM Pb resp.
0.1 mM Zn) had a stronger effect (more than twofold up-regu-
lation) than the higher concentrations (5 mM Pb resp. 1 mM
Zn; less than twofold up-regulation).
p032E08_ORF1 (peptide transporter): The protein putatively
encoded by p032A08_ORF1 contains 12 predicted TM domains,
from which 10 are unambiguously predicted by the TMHMM
server, whereas TM-helices 1 and 2 are only predicted with
low probability. p032A08_ORF1 belongs to the proton-depen-
dent oligopeptide transporter (POT) family (TC2.A.17.) within
the MFS. Not all POT family members necessarily transport
peptides, but also can transport nitrate (Zhou et al. 1998), nitrite
(Sugiura et al. 2007), histidine (Frommer et al. 1994; Zhou et al.
1998) or antibiotics (Wenzel et al. 1995).
S. pombe contains two peptide transporters: the glutathione
transporter Pgt1 (Thakur et al. 2008) and the PTR family pep-
tide transporter Ptr2 (SPBC13A2.04c). Expression levels of
pgt1 reach a maximum (6.2-fold) after 15 min exposure to
Cd, but decline to lower levels (1.8-fold) after 60 min (Chen
et al. 2003). The uncharacterized peptide transporter ptr2, on
the other hand, shows only marginal levels of up-regulation
(1.2-fold) after 15 min and down-regulation (0.8-fold) after
60 min (Chen et al. 2003). The C. finlandica putative peptide
transporter p032A08_ORF1 shows higher similarity to SpPtr2
than to SpPgt1.
p039H05_ORF2 (hypothetical proteins): p039H05_ORF2 is
predicted to be an extracellular protein with a cleavable signal
peptide. Highest homology is found to a predicted protein
from the ectomycorrhizal basidiomycete Laccaria bicolor
(XP_001878943, E value 10�27) (Martin et al. 2008), whereas all
other proteins in the database have E values of 3� 10�4 or
higher. The protein family represented by p039H05_ORF2
and L. bicolor XP_001878943 seems to be absent from the
adophora finlandica. Only the regulated genes are shown in
ntal Fig 1. Classes I-V refer to transcriptional patterns as
ponse to Cd, Pb and Zn, resp. Graphs on the right show rel-
-metal treatment set to 100 %. Expression of gpdA and tefA
1386 M. Gorfer et al.
majority of fungi and is maybe restricted to fungi with the po-
tential to form ECM. p039H05_ORF2 is slightly down-regulated
by Cd and more strongly up-regulated by both, Pb and Zn.
Both, up- and down-regulation show a dose response, with
higher metal concentrations exerting stronger effects.
Class Vp075G11_ORF2 (b-carbonic anhydrase, clade D): p075G11_ORF2
is predicted to be a b-carbonic anhydrase (bCA), the main role of
which is maintenance of internal pH and CO2/bicarbonate bal-
ances required for biosynthetic reactions (Zimmerman & Ferry
2008). Whereas the C. finlandica bCA is down-regulated upon ex-
posure to Cd, Pb or Zn, the only carbonic anhydrase (CA, also
from the b-family) found in S. pombe is up-regulated more than
twofold in response to Cd (Chen et al. 2003). Whether C. finlandica
contains other CAs is currently not known. Differences in Cd re-
sponse could also arise from differences in experimental condi-
tions. In S. pombe responses to Cd were assayed after 150 and 600
of exposure, whereas in C. finlandica responses were assayed af-
ter two days of exposure. The exact role of CA-regulation in re-
sponse to HMs is currently unknown.
p075G11_ORF3 (centaurin b): p075G11_ORF2 is composed of
an N-terminal BAR (Bin–Amphiphysin–Rvs) domain, a central
PH (pleckstrin-homology) domain and a C-terminal ArfGAP
(ADP ribosylation factor GTPase activating protein), an architec-
ture also found in centaurin b (Nie & Randazzo 2006). BAR do-
mains are dimerization, membrane binding, and membrane
curvature sensing modules found in many protein families (Pe-
ter etal. 2004);PHdomains show weakand nonspecificaffinity to
phosphosinositides and are frequently found among others in
proteins involved in cell signaling and cytoskeleton rearrange-
ment (Lemmon & Ferguson 2000); ArfGAPs are GTPase activat-
ing proteins of Arf and are involved in membrane trafficking
(Nie & Randazzo 2006; Randazzo et al. 2007). Disruption of the
orthologousgeneinS.pombe (cnt5),whichshowsthe same archi-
tecture as p075G11_ORF2, causes hypersensitivity to Cd and ar-
senic (As), with a more pronounced effect on Cd-sensitivity in
liquid culture and on As-sensitivity on plates (Vashisht et al.
2008). No Cd-dependent regulation was found at the transcrip-
tional level for S. pombe cnt5 (Chen et al. 2003; Vashisht et al.
2008). In contrast to these findings, the C. finlandica centaurin
b orthologue p075G11_ORF2 is strongly down-regulated at the
transcriptional level upon exposure to Cd, Pb or Zn. The exis-
tence of a C. finlandica paralogue with an inverse transcription
pattern can not be excluded, but the closely related helotialean
fungi Botryotinia fuckeliana and Sclerotinia sclerotiorum only have
one copy of a centaurin b orthologue in their genomes among
a total number of four respectively five ArfGAPs.
p085C08_ORF3 (GNAT-family acetyltransferase): p085C
08_ORF3 potentially encodes an acetyltransferase from the
GNAT-family (Gcn5-related N-acetyltransferases), which in-
cludes enzymes acetylating not only histone proteins but
also aminoglycoside-antibiotics, the mycothiol precursor, se-
rotonin and glucosamine-6-phosphate. Besides, longer acyl
groups can also be transferred by GNAT-family members, as
exemplified by the protein a-N-myristoyltransferase (Vetting
et al. 2005). In p085C08_ORF3 the GNAT domain is only par-
tially conserved. Probably, p085C08_ORF3 is involved in sec-
ondary metabolism together with an enzyme encoded by the
immediate downstream ORF p085C08_ORF2 (see Fig 2),
a protein related to phytanoyl-CoA-dioxygenases. Similar to
the GNAT domain in p085C08_ORF3, the phytanoyl-CoA-diox-
ygenase domain in p085C08_ORF2 is only partially conserved.
Proteins with phytanoyl-CoA-dioxygenase domains can be in-
volved in secondary metabolite production like the Streptomy-
ces lavendulae MmcH, which is necessary for mitomycin
synthesis (Mao et al. 1999) or the Fusarium verticillioides
Fum3 p, which is necessary for fumonisin biosynthesis (Ding
et al. 2004). In contrast to p085C08_ORF3, no HM dependent
regulation could be found for p085C08_ORF2.
Secondary metabolite production with antifungal activity
against Phialocephala fortinii Q104 (Gorfer et al. 2007) could be
observed for C. finlandica PRF15 (unpublished results).
Concluding remarks
In the present study, a genomic microarray for the root associ-
ated ascomycete Cadophora finlandica is presented. The utility of
the array for identification of regulated genes was demon-
strated in an attempt to elucidate the HM response of C. finland-
ica in axenic cultures. In future experiments the genomic array
can be used for the identification of regulated genes in complex
environments, as they are found e.g. in soil or in symbiotic re-
lationships. Cross-reactions from closely related organisms in
soil do not impose a major problem: Since data have to be con-
firmed by other methods, the necessary discrimination be-
tween true C. finlandica genes and highly homologous genes
from closely related fungi can be achieved e.g. by qPCR analy-
ses. The genomic array should therefore mainly be understood
as a tool for the cloning of differentially regulated genes and
not as a tool for transcriptome analysis.
The first set of HM regulated genes in C. finlandica shows
a high number of genes predicted to encode extracellular or
plasma membrane proteins. The majority of HM defence
activities in C. finlandica is therefore thought to take place
outside of the cell. Many of the newly identified HM regu-
lated genes encode proteins of unknown functions or pro-
teins with no established roles in HM detoxification. The
availability of a transformation and gene tagging system
for C. finlandica (Gorfer et al. 2007) as well as the availability
of an A. nidulans selectable marker on the plasmid back-
bone will be helpful in the future for the elucidation of
the functions that the newly identified C. finlandica HM-reg-
ulated proteins play.
Acknowledgements
We are grateful to Sylvia Fluch and Michael Stierschneider
from the ARC for assistance in microarray development.
This work was supported by grants LS149 (GENOMETALIX)
and LS0536 (NITROGENOM) from the Vienna Science and
Technology Fund (WWTF) to JS.
Supplementary information
Supplementary data associated with this article can be found
in the online version, at doi:10.1016/j.mycres.2009.09.005.
C. finlandica genomic array 1387
r e f e r e n c e s
Al-Lahham A, Rohde V, Heim P, Leuchter R, Veeck J,Wunderlich C, Wolf K, Zimmermann M, 1999. Biosynthesis ofphytochelatins in the fission yeast. Phytochelatin synthesis:a second role for the glutathione synthetase gene of Schizo-saccharomyces pombe. Yeast 15: 385–396.
Anonymus, 2005. The European EnvironmentdState and Outlook2005. European Environment Agency, Copenhagen.
Bae W, Chen X, 2004. Proteomic study for the cellular responses toCd2þ in Schizosaccharomyces pombe through amino acid-codedmass tagging and liquid chromatography tandem mass spec-trometry. Molecular & Cellular Proteomics 3: 596–607.
Banerjee S, Flores-Rozas H, 2005. Cadmium inhibits mismatchrepair by blocking the ATPase activity of the MSH2–MSH6complex. Nucleic Acids Research 33: 1410–1419.
Bara MT, Lima AL, Ulhoa CJ, 2003. Purification and characteriza-tion of an exo-beta-1,3-glucanase produced by Trichodermaasperellum. FEMS Microbiology Letters 219: 81–85.
Bellion M, Courbot M, Jacob C, Blaudez D, Chalot M, 2006. Extra-cellular and cellular mechanisms sustaining metal tolerance inectomycorrhizal fungi. FEMS Microbiology Letters 254: 173–181.
Bellion M, Courbot M, Jacob C, Guinet F, Blaudez D, Chalot M,2007. Metal induction of a Paxillus involutus metallothioneinand its heterologous expression in Hebeloma cylindrosporum.New Phytologist 174: 151–158.
Bendtsen JD, Nielsen H, von Heijne G, Brunak S, 2004. Improvedprediction of signal peptides: SignalP 3.0. Journal of MolecularBiology 340: 783–795.
Berger H, Basheer A, Bock S, Reyes-Dominguez Y, Dalik T,Altmann F, Strauss J, 2008. Dissecting individual steps of ni-trogen transcription factor cooperation in the Aspergillus ni-dulans nitrate cluster. Molecular Microbiology 69: 1385–1398.
Bhanoori M, Venkateswerlu G, 2000. In vivo chitin-cadmiumcomplexation in cell wall of Neurospora crassa. Biochimica etBiophysica Acta 1523: 21–28.
Birnboim HC, Doly J, 1979. A rapid alkaline extraction procedurefor screening recombinant plasmid DNA. Nucleic Acids Research7: 1513–1523.
Chen D, Toone WM, Mata J, Lyne R, Burns G, Kivinen K, Brazma A,Jones N, Bahler J, 2003. Global transcriptional responses offission yeast to environmental stress. Molecular Biology of theCell 14: 214–229.
Church GM, Gilbert W, 1984. Genomic sequencing. Proceedings ofthe National Academy of Sciences of the United States of America 81:1991–1995.
Coblenz A, Wolf K, 1994. The role of glutathione biosynthesis inheavy metal resistance in the fission yeast Schizosaccharomycespombe. FEMS Microbiology Reviews 14: 303–308.
Ding Y, Bojja RS, Du L, 2004. Fum3p, a 2-ketoglutarate-dependentdioxygenase required for C-5 hydroxylation of fumonisins inFusarium verticillioides. Applied and Environmental Microbiology70: 1931–1934.
Dos Santos Utmazian MN, Schweiger P, Sommer P, Gorfer M,Strauss J, Wenzel WW, 2007. Influence of Cadophora finlandicaand other microbial treatments on cadmium and zinc uptakein willows grown on polluted soil. Plant, Soil and Environment53: 158–166.
Edelmann SE, Staben C, 1994. A statistical analysis of sequencefeatures within genes from Neurospora crassa. ExperimentalMycology 18: 70–81.
Enjalbert B, Smith DA, Cornell MJ, Alam I, Nicholls S, Brown AJP,Quinn J, 2006. Role of the Hog1 stress-activated protein ki-nase in the global transcriptional response to stress in thefungal pathogen Candida albicans. Molecular Biology of the Cell17: 1018–1032.
Fauchon M, Lagniel G, Aude JC, Lombardia L, Soularue P, Petat C,Marguerie G, Sentenac A, Werner M, Labarre J, 2002. Sulfursparing in the yeast proteome in response to sulfur demand.Molecular Cell 9: 713–723.
Fernandes L, Rodrigues-Pousada C, Struhl K, 1997. Yap, a novelfamily of eight bZIP proteins in Saccharomyces cerevisiae withdistinct biological functions. Molecular and Cellular Biology 17:6982–6993.
Fogarty RV, Tobin JM, 1996. Fungal melanins and their interac-tions with metals. Enzyme and Microbial Technology 19: 311–317.
Fomina M, Hillier S, Charnock JM, Melville K, Alexander IJ,Gadd GM, 2005. Role of oxalic acid overexcretion in transfor-mations of toxic metal minerals by Beauveria caledonica. Appliedand Environmental Microbiology 71: 371–381.
Frommer WB, Hummel S, Rentsch D, 1994. Cloning of an Arabi-dopsis histidine transporting protein related to nitrate andpeptide transporters. FEBS Letters 347: 185–189.
Gadd GM, 2007. Geomycology: biogeochemical transformations ofrocks, minerals, metals and radionuclides by fungi, biowea-thering and bioremediation. Mycological Research 111: 3–49.
Gasch AP, Spellman PT, Kao CM, Carmel-Harel O, Eisen MB,Storz G, Botstein D, Brown PO, 2000. Genomic expressionprograms in the response of yeast cells to environmentalchanges. Molecular Biology of the Cell 11: 4241–4257.
Gebhart D, Bahrami AK, Sil A, 2006. Identification of a copper-inducible promoter for use in ectopic expression in the fungalpathogen Histoplasma capsulatum. Eukaryotic Cell 5: 935–944.
Goller SP, Gorfer M, Kubicek CP, 1998. Trichoderma reesei prs12encodes a stress- and unfolded-protein-response-inducibleregulatory subunit of the fungal 26S proteasome. CurrentGenetics 33: 284–290.
Gonzalez-Chavez MC, Carrillo-Gonzalez R, Wright SF, Nichols KA,2004. The role of glomalin, a protein produced by arbuscularmycorrhizal fungi, in sequestering potentially toxic elements.Environmental Pollution 130: 317–323.
Gorfer M, Klaubauf S, Bandian D, Strauss J, 2007. Cadophora fin-landica and Phialocephala fortinii: Agrobacterium-mediatedtransformation and functional GFP-expression. MycologicalResearch 111: 850–855.
Halliwell B, Gutteridge J, 1999. Free Radicals in Biology and Medicine.Oxford Science Publications, New York.
Harrington TC, McNew DL, 2003. Phylogenetic analysis places thePhialophora-like anamorph genus Cadophora in the Helotiales.Mycotaxon 87: 141–152.
Hirata D, Yano K, Miyakawa T, 1994. Stress-induced transcriptionalactivation mediated by YAP1 and YAP2 genes that encode theJun family of transcriptional activators in Saccharomyces cerevi-siae. Molecular and General Genetics 242: 250–256.
Hwang L, Hocking-Murray D, Bahrami AK, Andersson M, Rine J,Sil A, 2003. Identifying phase-specific genes in the fungalpathogen Histoplasma capsulatum using a genomic shotgunmicroarray. Molecular Biology of the Cell 14: 2314–2326.
Ivey FD, Magee DM, Woitaske MD, Johnston SA, Cox RA, 2003.Identification of a protective antigen of Coccidioides immitis byexpression library immunization. Vaccine 21: 4359–4367.
Jacob C, Courbot M, Brun A, Steinman HM, Jacquot JP, Botton B,Chalot M, 2001. Molecular cloning, characterization and reg-ulation by cadmium of a superoxide dismutase from the ec-tomycorrhizal fungus Paxillus involutus. European Journal ofBiochemistry 268: 3223–3232.
Jacob C, Courbot M, Martin F, Brun A, Chalot M, 2004. Transcrip-tomic responses to cadmium in the ectomycorrhizal fungusPaxillus involutus. FEBS Letters 576: 423–427.
Jekosch K, Kuck U, 1999. Codon bias in the ß-lactam producerAcremonium chrysogenum. Fungal Genetics Newsletter 46: 11–13.
Jungmann J, Reins HA, Schobert C, Jentsch S, 1993. Resistance tocadmium mediated by ubiquitin-dependent proteolysis. Na-ture 361: 369–371.
1388 M. Gorfer et al.
Lemmon MA, Ferguson KM, 2000. Signal-dependent membranetargeting by pleckstrin homology (PH) domains. BiochemicalJournal 350 (Pt 1): 1–18.
Li ZS, Lu YP, Zhen RG, Szczypka M, Thiele DJ, Rea PA, 1997. A newpathway for vacuolar cadmium sequestration in Saccharomy-ces cerevisiae: YCF1-catalyzed transport of bis(glutathionato)-cadmium. Proceedings of the National Academy of Sciences of theUnited States of America 94: 42–47.
Lloyd AT, Sharp PM, 1991. Codon usage in Aspergillus nidulans.Molecular and General Genetics 230: 288–294.
Mao Y, Varoglu M, Sherman DH, 1999. Molecular characterizationand analysis of the biosynthetic gene cluster for the antitumorantibiotic mitomycin C from Streptomyces lavendulae NRRL2564. Chemistry & Biology 6: 251–263.
Markham NR, Zuker M, 2005. DINAMelt web server for nucleicacid melting prediction. Nucleic Acids Research 33: W577–581.
Martin F, Aerts A, Ahren D, Brun A, Danchin EG, Duchaussoy F,Gibon J, Kohler A, Lindquist E, Pereda V, Salamov A,Shapiro HJ, Wuyts J, Blaudez D, Buee M, Brokstein P,Canback B, Cohen D, Courty PE, Coutinho PM, Delaruelle C,Detter JC, Deveau A, DiFazio S, Duplessis S, Fraissinet-Tachet L, Lucic E, Frey-Klett P, Fourrey C, Feussner I, Gay G,Grimwood J, Hoegger PJ, Jain P, Kilaru S, Labbe J, Lin YC,Legue V, Le Tacon F, Marmeisse R, Melayah D, Montanini B,Muratet M, Nehls U, Niculita-Hirzel H, Oudot-Le Secq MP,Peter M, Quesneville H, Rajashekar B, Reich M, Rouhier N,Schmutz J, Yin T, Chalot M, Henrissat B, Kues U, Lucas S, Vande Peer Y, Podila GK, Polle A, Pukkila PJ, Richardson PM,Rouze P, Sanders IR, Stajich JE, Tunlid A, Tuskan G,Grigoriev IV, 2008. The genome of Laccaria bicolor provides in-sights into mycorrhizal symbiosis. Nature 452: 88–92.
Martinez-Pastor MT, Marchler G, Schuller C, Marchler-Bauer A,Ruis H, Estruch F, 1996. The Saccharomyces cerevisiae zinc fingerproteins Msn2p and Msn4p are required for transcriptionalinduction through the stress response element (STRE). TheEMBO Journal 15: 2227–2235.
Meharg AA, 2003. The mechanistic basis of interactions betweenmycorrhizal associations and toxic metal cations. MycologicalResearch 107: 1253–1265.
Nie Z, Randazzo PA, 2006. Arf GAPs and membrane traffic. Journalof Cell Science 119: 1203–1211.
Nittler MP, Hocking-Murray D, Foo CK, Sil A, 2005. Identificationof Histoplasma capsulatum transcripts induced in response toreactive nitrogen species. Molecular Biology of the Cell 16:4792–4813.
O’Connell MJ, Osmani AH, Morris NR, Osmani SA, 1992. An extracopy of nimEcyclinB elevates pre-MPF levels and partially sup-presses mutation of nimTcdc25 in Aspergillus nidulans. The EMBOJournal 11: 2139–2149.
Ortiz DF, Kreppel L, Speiser DM, Scheel G, McDonald G, Ow DW,1992. Heavy metal tolerance in the fission yeast requires anATP-binding cassette-type vacuolar membrane transporter.The EMBO Journal 11: 3491–3499.
Peter BJ, Kent HM, Mills IG, Vallis Y, Butler PJ, Evans PR,McMahon HT, 2004. BAR domains as sensors of membranecurvature: the amphiphysin BAR structure. Science 303:495–499.
Plocke DJ, Kagi JH, 1992. Spectral characteristics of cadmium-containing phytochelatin complexes isolated from Schizosac-charomyces pombe. European Journal of Biochemistry 207: 201–205.
R Development Core Team. 2005. R Foundation for StatisticalComputing, Vienna, Austria.
Randazzo PA, Inoue H, Bharti S, 2007. Arf GAPs as regulators ofthe actin cytoskeleton. Biology of the Cell 99: 583–600.
Ren Q, Chen K, Paulsen IT, 2007. TransportDB: a comprehensivedatabase resource for cytoplasmic membrane transport sys-tems and outer membrane channels. Nucleic Acids Research 35:D274–279.
Riggle PJ, Kumamoto CA, 2000. Role of a Candida albicans P1-typeATPase in resistance to copper and silver ion toxicity. TheJournal of Bacteriology 182: 4899–4905.
Rodrigues-Pousada CA, Nevitt T, Menezes R, Azevedo D, Pereira J,Amaral C, 2004. Yeast activator proteins and stress response:an overview. FEBS Letters 567: 80–85.
Rozen S, Skaletsky HJ, 2000. Primer3 on the WWW for generalusers and for biologist programmers. In: Krawetz S, Misener S(eds), Bioinformatics Methods and Protocols: methods in molecularbiology. Humana Press, Totowa, NJ, pp. 365–386.
Sa-Correia I, dos Santos SC, Teixeira MC, Cabrito TR, Mira NP,2009. Drug:Hþ antiporters in chemical stress response inyeast. Trends in Microbiology 17: 22–31.
Sambrook J, Russell D, 2001. Molecular Cloning, A Laboratory Man-ual, 3rd edn. Cold Spring Harbor Laboratory Press, USA.
Smyth GK, Speed T, 2003. Normalization of cDNA microarraydata. Methods 31: 265–273.
Stuart GW, Searle PF, Palmiter RD, 1985. Identification of multiplemetal regulatory elements in mouse metallothionein-I pro-moter by assaying synthetic sequences. Nature 317: 828–831.
Sugiura M, Georgescu MN, Takahashi M, 2007. A nitrite trans-porter associated with nitrite uptake by higher plant chloro-plasts. Plant and Cell Physiology 48: 1022–1035.
Thakur A, Kaur J, Bachhawat AK, 2008. Pgt1, a glutathionetransporter from the fission yeast Schizosaccharomyces pombe.FEMS Yeast Research 8: 916–929.
Vallee BL, Ulmer DD, 1972. Biochemical effects of mercury, cad-mium, and lead. Annual Review of Biochemistry 41: 91–128.
Vallino M, Drogo V, Abba’ S, Perotto S, 2005. Gene expression ofthe ericoid mycorrhizal fungus Oidiodendron maius in thepresence of high zinc concentrations. Mycorrhiza 15: 333–344.
Vallino M, Martino E, Boella F, Murat C, Chiapello M, Perotto S,2009. Cu, Zn superoxide dismutase and zinc stress in themetal-tolerant ericoid mycorrhizal fungus Oidiodendron maiusZn. FEMS Microbiology Letters 293: 48–57.
Vanden Wymelenberg A, Sabat G, Mozuch M, Kersten PJ, Cullen D,Blanchette RA, 2006. Structure, organization, and transcrip-tional regulation of a family of copper radical oxidase genes inthe lignin-degrading basidiomycete Phanerochaete chrysospo-rium. Applied and Environmental Microbiology 72: 4871–4877.
Vashisht AA, Kennedy PJ, Russell P, 2008. Centaurin-like proteinCnt5 contributes to arsenic and cadmium resistance in fissionyeast. FEMS Yeast Research 9: 257–269.
Vetting MW, de Carvalho LP, Yu M, Hegde SS, Magnet S,Roderick SL, Blanchard JS, 2005. Structure and functions of theGNAT superfamily of acetyltransferases. Archives of Biochem-istry and Biophysics 433: 212–226.
Vralstad T, Myhre E, Schumacher T, 2002. Molecular diversity andphylogenetic affinities of symbiotic root-associated ascomy-cetes of the Helotiales in burnt and metal polluted habitats.New Phytologist 155: 131–148.
Weissman Z, Berdicevsky I, Cavari BZ, Kornitzer D, 2000. The highcopper tolerance of Candida albicans is mediated by a P-typeATPase. Proceedings of the National Academy of Sciences of theUnited States of America 97: 3520–3525.
Wenzel U, Thwaites DT, Daniel H, 1995. Stereoselective uptake ofbeta-lactam antibiotics by the intestinal peptide transporter.British Journal of Pharmacology 116: 3021–3027.
Wenzel WW, Jockwer F, 1999. Accumulation of heavy metals inplants grown on mineralised soils of the Austrian Alps. Envi-ronmental Pollution 104: 145–155.
Zhou JJ, Theodoulou FL, Muldin I, Ingemarsson B, Miller AJ, 1998.Cloning and functional characterization of a Brassica napustransporter that is able to transport nitrate and histidine.Journal of Biological Chemistry 273: 12017–12023.
Zimmerman SA, Ferry JG, 2008. The beta and gamma classesof carbonic anhydrase. Current Pharmaceutical Design 14:716–721.