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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

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