Matingtype orthologous genes in the primarily homothallic Moniliophthora perniciosa, the causal agent of Witches' Broom Disease in cacao

442

Journal of Basic Microbiology 2010, 50, 442 – 451

www.jbm-journal.com

Research Paper

Mating-type orthologous genes in the primarily homothallic
Moniliophthora perniciosa, the causal agent
of Witches’ Broom Disease in cacao

Ursula Kües and Mónica Navarro-González

Division of Molecular Wood Biotechnology and Technical Mycology, Büsgen-Institute,
Georg-August-University Göttingen, Göttingen, Germany

The cacao-pathogenic Moniliophthora perniciosa C-biotype is a primarily homothallic Agari-
comycete of which the genome has recently become available. Searching of the genome
sequence with mating type proteins from other basidiomycetes detected one or possibly two
potential genes for HD1 homeodomain transcription factors, 7 or possibly 8 genes for potential
pheromone receptors and five genes for putative pheromone precursors. Apparently, the
fungus possesses gene functions encoded in the tetrapolar basidiomycetes in the A and B
mating loci, respectively. In the tetrapolar species, the A and B mating type genes govern
formation of clamp cells at hyphal septa of the dikaryon and their fusion with sub-apical cells
as well as mushroom production. The C-biotype forms fused clamp cells and also basidiocarps
on mycelia germinated from basidiospores and their development might be controlled by the
detected genes. It represents the first example of a primarily homothallic basidiomycete where
A- and B-mating-type-like genes were found. Various strategies are discussed as how self-
compatibility in presence of such genes can evolve. An A-mating-type like gene for an HD2
homeodomain transcription factor is, however, not included in the available sequence
representing estimated 69% coverage of the haploid genome but there are non-mating genes
for other homeodomain transcription factors of currently unknown function that are
conserved in basidiomycetes and also various ascomycetes.

Keywords: Mating types / Homeodomain transcription factors / Pheromones /

G protein-coupled pheromone receptors / Basidiomycete

Received: January 09, 2010, accepted: March 25, 2010

DOI 10.1002/jobm.201000013

Introduction

Mondego et al. [1] recently published a genome survey
of Moniliophthora perniciosa (Crinipellis perniciosa), a hemi-
biotrophic basidiomycete pathogenic to Theobroma ca-
cao. The genome was established in 1.9 × coverage from
size-selected fragmented genomic DNA of shot-gun-
cloned segments of about 2 kb in length. Analysis of
all reads (average length 550 bp) together allowed to
assemble 17,991 contigs, the largest of which had
25,364 bp. In addition, there were 7,065 singlets with

Correspondence: Prof. Dr. Ursula Kües, Division of Molecular Wood
Biotechnology and Technical Mycology, Büsgen-Institute, Georg-
August-University Göttingen, Büsgenweg 2
D-37077 Göttingen, Germany
E-mail: ukuees@gwdg.de
Phone: +49-551-397024

average lengths of 1,300 bp and 455  bp, respectively.
The total length of available sequence adds up to
27 Mbp. With an estimated haploid genome size of
39 Mbp, this represents 69% to the total genomic se-
quence. Using sequences from an EST library for train-
ing gene predictor programs such as AUGUSTUS, SNAP
and Genezilla, a total of 16,329 gene models were cre-
ated by computorial approaches. Many of these pre-
dicted genes are however incomplete due to the
generally short length of the contigs. Nevertheless,
M. perniciosa  gene models present good similarity to
genes from the also sequenced Agaricomycetes Copri-
nopsis cinerea, Laccaria bicolor, Phanerochaete chrysosporium,
Schizophyllum commune and Postia placenta.
The cacao-pathogenic M.

perniciosa is primarily

homothallic and non-outbreeding. Basidiospores ger-

Journal of Basic Microbiology 2010, 50, 442 – 451

Mating-type-like genes in Moniliophthora perniciosa 443

www.jbm-journal.com

minate into a mycelium that will develop clamp cells
within a few days upon germination [2]. M. perniciosa
infesting Theobroma species and the related malvaceous
genus  Herrania is known as C-biotype [3]. Related bio-
types have been described from other types of plants,
i.e. the S-biotype from solanaceous hosts, the L-biotype
from bignoniaceous lianas, and the H-biotype from the
malpighiaceous shrub Heteropterys acutifolia [4]. Within
this  M. perniciosa species complex, the H-biotype has
been redefined as Moniliophthora  (Crinipellis) brasiliensis
by means of morphometric (basidia, basidiospores) and
ITS data [5]. The relationship between the other bio-
types is less clear [6]. Whilst the S-bioptype is also pri-
marily homothallic, the L-biotype is tetrapolar hetero-
thallic and outcrossing with multiple A and B mating
type specificities. As in other tetrapolar Agaricomy-
cetes, the A and B mating type genes control dikaryon
formation and morphology including clamp cell forma-
tion and clamp cell fusion [2, 7].
  The mating type loci of Agaricomycetes are complex
in structure. The A  mating  type  loci  have  been  shown
to encode two types of homeodomain transcription
factors called HD1 and HD2 by one or more divergently
transcribed gene pairs. The products of the B mating
type loci are pheromones and pheromone receptors,
respectively, and there might be also groups of paralo-
gous genes within one locus. To be compatible in mat-
ing, two monokaryotic strains need to differ in their
mating type specificities and these are defined by the
specific alleles present at the two mating type loci.
Accordingly, only when differing at both mating type
loci, fusing sterile monokaryons can form a fertile di-
karyon on which fruiting bodies with basidiospores
arise. At the molecular level it was established that
HD1 and HD2 transcription factors from different
A mating type loci have to heterodimerize and that
pheromones and pheromone receptors from different
B mating type loci have to interact with each other in
order to control dikaryon formation and the related
sexual morphological changes [8].
  Genes related to the A and B mating type genes of
tetrapolar species are also present in bipolar Agarico-
mycetes [9–11] and in the possibly homothallic species
P. chrysosporium [12, 13] where some or all genes may
have lost their superior function in mating-type control
but not necessarily simultaneously their elementary
cellular functions. Mondego et al. [1] in their genome
survey of the primarily homothallic M. perniciosa C-bio-
type noticed seven different gene models whose prod-
ucts are similar to G-protein-coupled 7-transmembrane
pheromone receptors of the B mating type loci of the
tetrapolar  C. cinerea and Schizophyllum commune. Detec-

tion of genes for putative mating-type-like pheromones
and of genes resembling A mating type genes of Agari-
comycetes were not mentioned by the authors. Here,
we present an analysis of the M. perniciosa C-biotype of
potential genes related to A and B mating type genes of
other basidiomycetes.

Materials and methods

Sequences and sequence analysis
The complete genome of M. perniciosa FA553 as linked
on the ‘Blast with fungi’ genome page at NCBI
(http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi?or-
ganism=fungi) was subjected to tblastn searches using
protein sequences of interest. Hit contigs and singlets
were evaluated for defining coding regions of interest.
Where required, adjustments to published gene pre-
dictions were done. For protein comparisons, sequen-
ces were aligned using ClustalX (http://www-igbmc.u-
strasbg.fr/BioInfo/ClustalX/Top.html) and GeneDoc

version 2.6.002 (http://www.psc.edu/biomed/genedoc/).
Phylogenetic trees were calculated by the neighbour
joining method of MEGA version 4.0 [14].
The

5′ and 3′ truncated HD1 genes on CP02-PF-001-

002-G07-UC.R (NCBI GenBank accession number
ABRE01018067) and Contig1744 (ABRE01001637; com-
plement) cover the whole available sequences. The
putative homeodomain transcription factor gene on
Contig12913 (ABRE01012662; complement) is 3′ trun-
cated: join 1..166,221..342,396..703. For coding

regions on Contig11724 (ABRE01011481) join 1..121,
182..238,293..459.
  Coordinates of the 3′ truncated β-fg gene of Con-
tig14658 (ABRE01014396) are as follows: join 815..817,
866..920,982..1033. The 5′  half  of  the  mip gene as
present on Contig17178 (ABRE01016908; MPER_11852)
join to 1..342,395..723 on Contig14530 (ABRE01014268;
complement).
  Complete putative pheromone genes and complete or
partial genes for pheromone-like peptides of M. perni-
ciosa are found on the complementary strands of se-
quences as listed in Table 1.
  The complete pheromone receptor gene on Con-
tig15640 (ABRE01015374) of M. perniciosa was taken as
defined in NCBI GenBank. Predictions for other phero-
mone receptor genes were adjusted: For the 3′ trun-
cated gene on Contig14531 (ABRE01014269; comple-
ment) join 372..531,589..819,877..1014,1017..1128,
1200..1315,1370..1589. The prediction on Contig5948
(ABRE01005767; complement) is taken as suggested but
the start codon: the gene is 5′ and 3′ truncated and the

444

U. Kües and M. Navarro-González

Journal of Basic Microbiology 2010, 50, 442 – 451

www.jbm-journal.com

complete sequence 617..747 is part of an exon. For the
3´ truncated gene on Contig1742 (ABRE01001635; com-
plement) join 1..122,177..298,338..441. To obtain a
coding region for the 5´ truncated gene on Contig16294
(ABRE01016026; complement) join 1190..1348,
1424..1562,1616..2099,2156..2293,2365..2475. For read-
ing frames of the 5′ and 3′ truncated gene on CP02-S2-
032-503-E04-UC.F (ABRE01021646) join 1..70,119..487
and the 5′ and 3′ truncated gene on CP02-S2-041-311-
F08-EM.F (ABRE01022527; complement) join 1..189,
243..724. Furthermore, a putative pheromone receptor
gene starts at 1..93 on Contig12606 (ABRE01012359;
complement).
For coding regions of the putative clp1 of M. perni-
ciosa see 147..550 on CP02-S2-032-310-H05-UE.F
(ABRE01021382) and 23..175 on Contig4315
(ABRE01014189). Sequences resembling FTR1 of S. com-
mune are MPER_04098 from Contig7660
(ABRE01007448) and an N-terminal truncated protein
deduced from Contig16807 (ABRE01016537): join
1..22,79..233,295..420.

Results

Putative A mating type-like genes
The HD1 mating type proteins b1-2 (AAD33337) and
a1-2 (EAU92789) from paralogous A mating type genes
of C. cinerea [15] were used to search (blastn) the M. per-
niciosa genome. The 381 bp long CP02-PF-001-002-G07-
UC.R and the 533 bp long Contig1744 were hit due to
sequence conservation of the encoded HD1 homeodo-
main motifs. When using the complete sequence of
CP02-PF-001-002-G07-UC.R in a search (blastx) of the
complete NCBI database, HD1 mating type proteins a1-1
of L. bicolor and AαZ3 of S. commune came up as the most
similar proteins (Fig. 1A) amongst numerous other HD1
mating type proteins of different basidiomycete species.
The deduced product of Contig1744 is most similar to
the  C. cinerea HD1 mating type proteins b1-1 and d1-1
(Fig. 1B). Incidentally, the two M. perniciosa sequences
could come from the same gene overlapping by just one
nucleotide (A; position 703 in CP02-PF-001-002-G07-
UC.R; position 533 complement in Contig1744). Alter-
natively, as in the A mating type locus of other Agari-
comycetes containing pairs of duplicated HD1 and HD2
genes of paralogous function [10,13,16–18], paralogous
HD1-like genes could be present in M. perniciosa.
  Next, the HD2 mating type protein a2-1 of C. cinerea
(CAA56131) was used to tblastn (default setting) the
M. perniciosa genome. The 1390 bp long Contig12913
was hit by sequence similarities of the translated se-

quence to the HD2 homeodomain motif. A gene with at
least two introns was detected that translates into a
sequence with strong similarity to an uncharacterized
fungal transcription factor with a classical homeodo-
main. In a search (blastp of the NCBI database), a puta-
tive protein of L. bicolor was identified as most similar
followed by predicted proteins from allelic genes of a
sequenced dikaryon of the Agaricomycete P. placenta
and by a putative protein from C. cinerea. Searches
(tblastn on the ‘Blast with fungi genome’ page at NCBI)
indicated that the other basidiomycetes with submitted
genomes have also putative genes for related proteins,
as it is also true for a subset of the sequenced ascomy-
cetes (Fig. 1C). However, none of these genes are in-
cluded in any of the known fungal mating type loci and
have been shown to possess mating type function.
  Reducing the stringency (Expect 1000 in tblastn
search) using the C. cinerea a2-1 sequence, another gene
on Contig11724 was identified for a product related to
other fungal non-mating-type homeodomain transcrip-
tion factors such as a LIM-homeobox protein from the
Tremellomycete  Cryptococcus neoformans and the cell-
cycle regulator YOX1 of the ascomycete Saccharomyces
cerevisiae (Fig. 1D).
  Triggered by the obvious failure of detecting an HD2-
like gene in the M. perniciosa genome, other HD2 mating
type proteins from the basidiomycetes C. cinerea,  L. bi-
color, S. commune, C. neoformans and Ustilago maydis (Usti-
laginomycetes) were used (tblastn searches). However,
these also did not reveal an HD2-like mating type gene
in M. perniciosa.
The

A mating type locus in various Agaricomyetes is

directly flanked by the 3´ end of a conserved gene β-fg
for an unknown protein and, at the other end, by the
3′ end of a conserved gene mip for a mitochondrial
intermediate peptidase [10, 11, 13, 15–19]. Therefore,
tblastn searches of the M. perniciosa genome with β-fg
(EDR15176) and mip (EDR15793) from L. bicolor were
performed. Unfortunately, only the 5′ end of the
M. perniciosa β-fg gene was found on Contig14658. The 5′
half of the mip gene was detected on Contig17178 and a
continuing sequence on Contig14530. However, the 3′
end of mip was not identified that could help in defin-
ing the neighbouring gene. In conclusion, from the
currently available M. perniciosa  genome  sequence  we
are unable to deduce a complete A-mating-type-like
locus consisting of both HD1-like and HD2-like genes.

Genes for putative B mating type-like pheromone
receptors and pheromones
Of the seven genes for putative G-protein-coupled
7-transmembrane pheromone receptors found by Mon-

Journal of Basic Microbiology 2010, 50, 442 – 451

Mating-type-like genes in Moniliophthora perniciosa 445

www.jbm-journal.com

HKQ

NDE

GHA

SETLD

IDQSH

EVA

CDA

GQI

EFDHR

YPTTGN

YQT

QLD

EEA

SESVD

TLELA

QVA

AVEFR

NDL

CP02-PF-001-002A-G07-UC.R

a1-1

YQT

QLD

EEA

SESVD

TLELA

QVA

AVEFR

NDL

QTR

RAS

P---

QPS

TLEL

SDR

SYA

LAE

TQE

EDD

LSNDL

---

A Z3

CP02-PF-001-002-G07-UC.R

a1-1

A Z3

TPE

PSSSSRS

EPQPHQSTP

PPI

PYV

AAYEWLIQNLHNPYPT

LIDESLITQPG

GLE

V--------RSPRSSN

WLL

NLHNPYPT

AFEA

NDC

NMPPV

PPFI

YEWLLQ

LHNPYPS

A Z3

---

AFEA

NDC--------

NMPPV

PPFI

YEWLLQ

LHNPYPS

Contig1744

TAQ

QSGTSRK

WFID

RKRIGWN

IKK

RLH

GHEVDESE

KKRKEP

b1-1
d1-1

EVRT

IARQT

TSRKDIDAWFIDARRRIGWNEVRRK

NKR

DIV

AAS

TGPQ---

SIP

--AE

QVRS

IAKQTG

SRKDIDAWFIDARKRIGWNELRRR

NKR

NIV

AAT

HQGK---

EDLPC

QAQ

FAGI

HFVE

AGQ

LTP

IKEEV

QKH

---------L

ALPDH

IEL

FAGI

SRA

PSKLA

KLD

VKDMTP

LKEQLK

EAR

RKRE

ASTVGIINQ

Contig1744

b1-1

YGP

TIE

NDL

SAW

GRY

SKLA

KLD

VKDMTP

LKE

IKRD

ERRRE

ERQK---K

d1-1

ti 1744

LGHKHEAS

SSI

EVIV

PTPAP

KRRADN

EPE

VES

PSN

YPTPE

SPAS

ELL

PSFA

SDKLPSVG

KRR

LES

YPSPE

SPASS

SGAA

LSPV

VPV

IDL

SQCS

RRA

Contig1744

b1-1
d1-1

YPSPE

SPASS

SGAA

LSPV

VPV

IDL

SQCS

RRA

d1 1

---------------MCLPRFQ

DFAYNTFRHAQV

ESADYDAREH

TSH

DVL

---

MLTPLQ

SASS

PYYE

IDG

AAP

SEY

EPSSSQGAASTSNA

DAP

Contig12913

EAU83072

---------------MLTPLQ

SASS

PYYE

IDG

AAP

SEY

EPSSSQGAASTSNA

DAP---

MARHEPYPILDMRSRRASLRLD

AAVVHK

DEDR

AIT

YELDRVK

EDE

ELASGP

FRS

----------------MLTEI

SASTS

TTP

PDP

NAE

PAEGSP

APQH

---

EAU83072
EDR08756

EED85981

MLTEI

SASTS

TTP

PDP

NAE

PAEGSP

APQH

-----------MSSTDSPDQS

STT

PTTAPTT

SSS

PSI

NATTAP

AARRPQRK

T---

EED85981
EED55353

Contig12913

EAU83072
EDR08756

--------E

KRKR

RVT

EQL

HLE

PTAARRREI

LGM

ERQTQVWFQNRRAKAKVM

-----APKP

NHV

PPMV

RKEI

EEL

MSERQTQIWFQNRRAK

GSSAKSDTE

KRKR

RVT

EQLVHLE

PTA

RRREI

EQLGM

ERQTQIWFQNRRAKAKL

QEG

EDR08756

EED85981
EED55353

GSSAKSDTE

KRKR

RVT

EQLVHLE

PTA

RRREI

EQLGM

ERQTQIWFQNRRAKAKL

QEG

---PDGVKP

KRKR

RVS

EQLVHLE

PTAARRKEI

QLGMTERQTQIWFQNRRAKAKL

QAR

-----QQQKNN

QLV

PTAA

QEI

MTER

QIWFQNRRAK

KML

LPATLPHTKIVSNL

-------------------------

-D

NLIHE

DEI

VIPCT

EED55353

Contig12913

QQQKNN

QLV

PTAA

QEI

MTER

QIWFQNRRAK

KML

AAKPPTTRRS

PEAR

LTRSITPPPP--------------L

GFDFE

IIP

QSSVTEVSELSPD

PE----------------------L

NLIHE

IIPCT

QKAFAAPEAS

SEA

ICP

IHE

IPCT

EAU83072
EDR08756

EED85981

QKAFAAPEAS

SEA

D--------------------------ICP

IHE

IPCT

SIETGEGCD

IPE

MRQYLAMQFDPSKPGARDPFGRTGGYG

SGAYPSESTPSGKVV

HHFT

EED85981
EED55353

GSWRRIA

IAYLC

--Q

CLTWFV

ESS

GFKM

GTWKRVA

RCLTWYI

NNR

GFKM

VPY

Contig12913

EAU83072

GSWRRIA

TQTD

LLAYVC

RCLTWYI

AGY

GFKM

IPF

GSWKRIA

DLLAYV

HDTTGI

RCMSWFV

HCAGT

GFKM

GSWRRI

GQN

DLV

YSPE

CMTYYI

NNDSA

GYKI

EDR08756

EED85981
EED55353

GSWRRI

GQN--AM

DLV

YSPE

--A

CMTYYI

NNDSA

GYKI

EED55353

Contig11724

EDR

RCPA

ENSN

RTP

EER

ALARQVDMS

RSVQIWFQNKRQ

QT-

S--AQCMRS

PYD

EDR11530

EED80346

AAW45768

DAA

EER

ALAK

LDMS

RSVQIWFQNKRQ

QTN

SAHQ

MAN

MLEQ

EQR

ALAKE

RSV

IWFQNKRQ

RAK

PRH

YPFHDV

GKT

VLE

DIN

VQVWFQNRR

GLA

KEAEGQESKK

PENQE

AAW45768

CAA86628

VLE

DIN

VQVWFQNRR

GLA

KEAEGQESKK

PENQE

ILQ

ESCH

VQIWFQNKRQ

RQRIA

TIIQ

PPS

PS----------GN----------------Q

HVPR

SSP

EVHA

SPTL

TSS

H--

V-PRPQ

HGEPLLDDLSPSGYGGTPI---PILETPYMT

ITSRSH

SSS

H--

I--

IHTVRRAALLEQERGGSE

DQSSPPVGS

TTP

RASH

SPSL

EPM

PPLL

TPVH

TAR

Contig11724

EDR11530

EED80346

IHTVRRAALLEQERGGSE-------DQSSPPVGS

TTP

RASH

SPSL

EPM

PPLL

---D

TPVH

TAR

GTSSAIGPSPPLTDSAVSSSHFNLLPPPASVNMGRRA

LANGEAAK

EIFVA--

AAA

PPLDVHATPLASRVKADILRDGSSCSRSSSS

RPHH

LNRR

SST

PSI

S--

LTFH

NPQ

EED80346

AAW45768

CAA86628

PPLDVHATPLASRVKADILRDGSSCSRSSSS

RPHH

LNRR

SST

PSI

LTFH

NPQ

RYP

CAA86628

Contig11724

-W

GLL

SAPTFD

EDR11530

EED80346

AAW45768

GDAGANSP

PVK

AAW45768

CAA86628

Figure 1. Alignment of A. the translated protein sequence from CP02-PF-001-002-G07-UC.R of M. perniciosa and the corresponding
regions of the HD1 mating type proteins a1-1 of L. bicolor (EDR15177) and A

αZ3 of S. commune (AAB01369), B. the translated protein

sequence from Contig1744 and the corresponding sequence regions of the C. cinerea HD1 mating type proteins b1-1 (CAA44210) and
d1-1 (EAU92783),  C.  the deduced protein sequence of a gene on Contig12913 and the C-terminal truncated sequences of putative
non-mating-type homeodomain transcription factors of C. cinerea (EAU83072; for corrected gene sequence join AACS01000245:
32342..32679,32741..33003,33075..34367,34469..34473).  L. bicolor (EDR08756), P. placenta  (EED85981) and the ascomycetous mito-
sporic fungus Aspergillus flavus (EED55353) and D. the deduced protein sequence of a gene on Contig11724 and predicted proteins of
L. bicolor (EDR11530) and P. placenta (EED80346), the LIM-homeobox protein of C. neoformans (AAW45768) and YOX1 of S. cerevisiae
(CAA86628). For proteins of species other than M. perniciosa, only relevant protein regions are shown. Sequences belonging to
homeodomain motifs are underlined. The homeodomain motifs of the HD1 mating type proteins in A. and B. are shown incomplete due to
gene truncations of the respective M. perniciosa on the available contigs.

446

U. Kües and M. Navarro-González

Journal of Basic Microbiology 2010, 50, 442 – 451

www.jbm-journal.com

dego  et al. [1], only one is complete (on Contig15640).
Three of the other genes are 5′ and 3′ truncated, one
gene is 5′ truncated and two genes are 3′ truncated (see
Materials and methods). In addition, there might be
another pheromone receptor gene on Contig12606 by a
93 bp sequence that translates into a sequence highly
homologous to the N-termini of various basidiomy-
cetes’ pheromone receptors (not shown). On the DNA
level, this sequence has 77% identity to the first 93 bp
of the putative pheromone receptor gene on Con-
tig14531 and the deduced amino acid sequences have
83%  identity  and  87%  similarity  to  each  other.  It  is
however possible that the sequence on Contig12606
belongs to one of the four 5′ truncated genes and not to
a new, eighths pheromone receptor gene.
  A number of basidiomycetes have now been shown
to contain, other than pheromone receptor genes in the
mating type loci, elsewhere in the genome extra non-
mating-type pheromone receptor genes [9–11, 13, 17,
20]. However, phylogenetic evidence in C. cinerea and
L. bicolor indicates that pheromone receptor genes from
the  B mating type loci of Agaricomycetes are ortholo-
gous to each other whereas such genes outside the B
mating type locus are more distantly related to the
respective mating type genes [13]. In order to analyze
the potential pheromone receptors of M. perniciosa, we
performed a phylogenetic analysis with pheromone
receptors from other fungi (Fig. 2). Strikingly, the M.
perniciosa pheromone receptors from Contig14531 and
Contig5948 grouped specifically with two of the paralo-
gous  B mating type pheromone receptors of C. cinerea
(CcSTE3.1, CcSTE3.3) and their orthologous B mating
type pheromone receptors from L. bicolor (LbSTE3.1,
LBSTE3.3) coming from positional conserved genes [13,
21], whilst no sequence was particularly similar to gene
products from a third conserved location of the B mat-
ing type loci (CcSTE3.2a, CcSTE3.2b; LBSTE3.2). Other
M. perniciosa sequences clustered independently from
any foreign receptor sequence (Fig. 2).
All

basidiomycete

B mating type pheromone precur-

sor sequences from the NCBI database together with an
added symbol * at their C-terminal end (to find cor-
rectly positioned stop codons) were used in searches
(tblastn) of the M. perniciosa genome done at lowest
stringency (Expect 1000) by the generally very poor
conservation of fungal pheromone sequences (e.g. see
[13, 17, 21–24]). Two sequences for putative phero-
mones (on Contig12747 and Contig15613) indentified
by these searches are listed in Table 1. In addition to
genes for B mating-type pheromones, both C. cinerea and
L. bicolor possess large families of non-mating type
genes for conserved pheromone-like peptides [13, 25].

Potential genes for such pheromone-like peptides were
also detected in M. perniciosa (Table 1). Since pheromone
genes in other Agaricomycetes are clustered in groups
and linked to genes for pheromone receptor genes [13,
17, 21–24, 26, 27], all contigs with potential phero-
mone genes or putative pheromone receptor genes (see
below) were analyzed for presence of further phero-
mone genes but no more could be detected.

Genes for cellular functions overcoming mating-type
regulation in sexual development
Mutants in gene pcc1 in C. cinerea (BAA33018) have been
described that override the need of mating type genes
in creating mycelial dikaryon morphology and develop-
ing fruiting bodies. Pcc1 is an HMG box transcription
factor acting likely as repressor of sexual development
[28, 29]. In searches (tblastn) with Pcc1 against the
M. perniciosa genome, sequences were found for po-
tential HMG-box transcription factors [Contig11704
(ABRE01011461), Contig17486 (ABRE01017215), Con-
tig8803 (ABRE01008584), Contig10798 (ABRE01010559),
Contig15179 (ABRE01014916), CP02-S2-028-254-A08-
UE.F (ABRE01020867)]. In reciprocal searches against
the NCBI database (blastx), C. cinerea genes for potential
HMG box transcription factors came up, but similarity
was always restricted to the DNA binding motif permit-
ting no further conclusions on existence of pcc1 in
M. perniciosa.
  Clp1, as another regulator of sexual development in
C. cinerea, induces clamp cell production [30]. The
C. cinerea Clp1 sequence (EDR10292) identified two
sequences (CP02-S2-032-310-H05-UE.F: ABRE01021382;
Contig4315: ABRE01014189) that appear to carry the
5′ end and the 3′ end of the gene, respectively.
Another

gene,

frt1 (AAA74917), coding

for a regula-

tory protein with a Rex4-like motif, activates fruiting
when inserted ectopically into monokaryotic strains
of  S. commune [31, 32]. Two different proteins highly
similar to the first (47/63% identity/similarity) and the
second half of FTR1 (50/66% identity/similarity) are
apparently encoded on Contig7660 and Contig16807 of
M. perniciosa, respectively.

Discussion

Different biotypes of the fungal pathogen M. perniciosa
occur on shrubs in tropical countries. The heterothallic
L-biotype has two mating type loci with multiple alleles
and these were found to control clamp cell formation
and fusion, respectively [2, 7]. Mating type genes typical
of basidiomycetes are therefore expected for the tetra-

Journal of Basic Microbiology 2010, 50, 442 – 451

Mating-type-like genes in Moniliophthora perniciosa 447

www.jbm-journal.com

LbSTE3.3

CcSTE3.3

Contig5948

Contig14531

LbSTE3.1

CcSTE3.1

Contig15640

LbSTE3.4

CP02-S2-041-311-F08-EM.F

Contig16294

CP02-S2-032-503-E04-UC.F

CcSTE3 2151

LbSTE3.13

CcSTE3 7395

LbSTE3.9

CnSte3a

ScSte3p

LbSTE3.2

CcSTE3.2b

CcSTE3.2a

LbSTE3.6

CcSTE3 2153

CnSte3α

CnCpr2

LbSTE3.5

ScSte2p

0.2

B Gene Subgroup 3

B Gene Subgroup 1

B Gene Subgroup 2

LbSTE3.4

CcSTE3-2151

Contig15640

Contig1742

CcSTE3-7395

LbSTE3.9

LbSTE3.3

CcSTE3.3

Contig14531

LbSTE3.1

CcSTE3.1

LbSTE3.13

CnSte3α

LbSTE3.6

CcSTE3 2153

LbSTE3.5

CcSTE3.2b

LbSTE3.2

CcSTE3.2a

CnSte3a

CnCpr2

ScSte3p

ScSte2p

0.2

B Gene Subgroup 3

B Gene Subgroup 1

B Gene Subgroup 2

LbSTE3.3

CcSTE3.3

Contig5948

Contig14531

LbSTE3.1

CcSTE3.1

Contig15640

LbSTE3.4

CP02-S2-041-311-F08-EM.F

Contig16294

CP02-S2-032-503-E04-UC.F

CcSTE3 2151

LbSTE3.13

CcSTE3 7395

LbSTE3.9

CnSte3a

ScSte3p

LbSTE3.2

CcSTE3.2b

CcSTE3.2a

LbSTE3.6

CcSTE3 2153

CnSte3α

CnCpr2

LbSTE3.5

ScSte2p

0.2

B Gene Subgroup 3

B Gene Subgroup 1

B Gene Subgroup 2

LbSTE3.4

CcSTE3-2151

Contig15640

Contig1742

CcSTE3-7395

LbSTE3.9

LbSTE3.3

CcSTE3.3

Contig14531

LbSTE3.1

CcSTE3.1

LbSTE3.13

CnSte3α

LbSTE3.6

CcSTE3 2153

LbSTE3.5

CcSTE3.2b

LbSTE3.2

CcSTE3.2a

CnSte3a

CnCpr2

ScSte3p

ScSte2p

0.2

B Gene Subgroup 3

B Gene Subgroup 1

B Gene Subgroup 2

Figure 2. Phylogenetic analysis of pheromone receptors of the basidiomycetes M. perniciosa (marked by grey shading), C. cinerea
(EAU87370 = CcSTE3.1; corrected EAU87377, join complement AACS01000134: 162667..162895,162948..163392,163443..163580,
163633..163815,163875..164073 = CcSTE3.2a; corrected EAU87377, join complement AACS01000134: 161053..161509,161568..161705,
161760..161939,161999..162206 = CcSTE3.2b; EAU87378 = CcSTE3.3; corrected EAU87392: join complement AACS01000134:
209361..210490,210541..210663,210716..210812 = CcSTE3-2151; corrected EAU87394, join complement AACS01000134:

212458..213373,213434..213875,213932..214069,214121..214301,214363..214564 = CcSTE3-2153; EAU91360 = CcSTE3-7395),
L. bicolor (models were taken from http://genome.jgi-psf.org/Lacbi1/Lacbi1.home.html as given in [13]), C. neoformans (AAN75624 =
CnSte3a; CnSte3

α = XP_570116; CnCpr2 = AAW41941) and the two mating type pheromone receptors ScSte2p and ScSte3p of the

ascomycete S. cerevisiae (EEU04250, EEU09227). Paralogous B mating type pheromone receptors of C. cinerea and of L. bicolor are
presented in bold and labelled with the name of the paralogous subgroups they belong to [13]. Due to incomplete sequences of
M. perniciosa, the first 107 – 117 amino acids from the utmost N-terminal sequences (A) and more inner regions of the N-termini of 127 to
321 amino acids in length (B) were independently analyzed from proteins where available. Bootstrapping values (500 replications) above
50 are shown at tree branchings. The scale bar defines the number of nucleotide substitutions per site.

Table 1. Potential precursors for pheromones and pheromone-like peptides in M. perniciosa.

Accession

Contig/bp position

Deduced peptide sequence

ABRE01012499 Contig12747/1295-1113

MVIRPFSRRWEMVSQPARRVMSYGQGKRVVTHRCRLNLHTTP

CDYSRYEIFLRRSLCLIS

ABRE01015347 Contig15613/614-366

MDSFENFDFLALASDESPIHNPFASFSTSLSEDASEVPSAAL

FNIESCRSRSFELQTDSQSLPTNHERAEAAAVHGGFCVIA

ABRE01010055 Contig10289/424-311

MDSFTTITSSVEDITVSLPVPVDEELKGGGWAYCVIA

ABRE01004489 Contig4617/721-650

…VISLPVPVDEEQNGGGWAYCVIA

ABRE01019551 CP02-S2-000-167-C03-UC.F/471-415

…IPINEEQQGGGWCYCVIA

i. Predicted N-terminal recognition sites (charged doublets) and the C-terminal CAAX-motifs (cysteine, aliphatic amino acid,

aliphatic amino acid, any amino acid) typical for basidiomycete pheromones [8] are underlined. ii. The potential genes for

pheromone-like proteins on Contig4617 and CP02-S2-000-167-C03-UC.F are incomplete by 5′ truncations.

448

U. Kües and M. Navarro-González

Journal of Basic Microbiology 2010, 50, 442 – 451

www.jbm-journal.com

polar L-biotype. The primarily homothallic C-biotype [2]
has no obvious need for such genes acting in mating.
Nevertheless, this study and the earlier work by Mon-
dego et al. [1] indicate that the fungus indeed possesses
genes orthologous to mating type genes of heterothallic
basidiomycetes. Markedly, post-mating functions being
in heterothallics under control of the mating type
genes such as nuclear pairing, synchronized nuclear
division, clamp cell production and also fruiting [33–
36] are performed in the C-biotype [2]. Moreover, the
disposability of self-compatibility does not essentially
block the overall possibility of mating to other strains
and the employment of mating type genes as demon-
strated by crosses between self-compatible and self-
sterile strains of C. cinerea [37].
From nutritionally forced crosses of primarily
homothallic and bipolar heterothallic forms of the
Agaricomycete  Sistostrema brinkmannii resulting in a
new mating type within a self-sterile offspring, Lemke
[38] suggested that primarily homothallic organisms
will have a cryptic or masked mating type locus. Ull-
rich and Raper [39] put forward the duplication of
the single mating type locus and inclusion of compati-
ble alleles in one genome as one possible explanation
for creating in S. brinkmannii a primary homothallic
form from the bipolar situation. Experimental indica-
tions for this scenario is not available for any basidio-
mycete up to now, and the lack of evidence for ane-
uploidy or insertional translocation in progenies of
crosses between primarily homothallic and bipolar
heterothallic isolates of S. brinkmannii might be taken
as counter-argument [39]. Conversion from heterothal-
lism to homothallism by mutation of genes respon-
sible for mating type specificity in one or more nuclei
within the mycelium was regarded as improbable,
since extensive mutagenesis programs in various basi-
diomycetes all failed to create such mutants [39]. Non-
etheless, switching events in mating types with gen-
eration of new specificities have subsequently been
documented in certain monokaryotic strains of the
tetrapolar Agaricomycete Agrocybe aegerita. Progenies
with new mating types, however, germinated into
monokaryons being self-sterile up to another event of
mating type switching [40]. Therefore, mating type
switching as such does not explain the occurrence of
primary homothallism within the Agaricomycetes.
Moreover, deletion of all mating type genes from
a haploid genome as an alternative hypothesis for
primary homothallism [39] can be ruled out since in
C. cinerea a mutant missing functional A mating type
genes and in S. commune a strain without the typical B
mating type locus are available which both have the

normal self-sterile monokaryon phenotypes [23, 26,
41].
  Rare mutations to constitutive self-fertility are re-
ported in C. cinerea [42, 43] as well as in S. commune
[44]. For the A mating type locus in C. cinerea, self-
compatibility was shown to be caused by chromoso-
mal mutations fusing together parts of HD2 and HD1
genes. The resulting chimeric genes encode products
that overcome the need for the normal heterodimeriza-
tion between independent HD1 and HD2 proteins
coming  from  different  mating  types  [41,  43].  Here,
we have found evidence for (a) putative HD1-like gene(s)
in the M. perniciosa C-biotype. HD2-like genes were miss-
ing in the available genome sequences but might have
simply escaped sequencing so far. In C. cinerea, the
homeodomains of the HD2 proteins are required for
the regulatory functions of active transcription factor
heterodimers as well as of the chimeric HD2-HD1
mutant transcription factors [45, 46]. A potential loss
of  HD2 genes thus would neither explain self-
compatibility nor expression of dikaryon-specific mor-
phological phenotypes. The available M. perniciosa se-
quences for (a) HD1-like gene(s) are not complete due
to the short lengths of Contig1744 and CP02-PF-001-
002-G07-UC.R (Fig. 1). It currently cannot be ruled
out that the sequences are part of chimeric HD2-HD1
genes mediating self-compatibility as described in
the constitutively activated A mating type mutants
of  C. cinerea [41, 43]. Homothallism however could
also easily be achieved if compatible HD1-like and HD2-
like genes are present in the same nucleus that en-
code proteins able to heterodimerize to an active
transcription factor complex allowing full function
in regulation of dikaryon maintenance and fruiting
events.
Mutations

C. cinerea and S. commune leading to self-

compatibility in B and subsequent constitutive expres-
sion of B-mating-type-controlled processes either ren-
dered a pheromone receptor constitutive or changed
the specificity of a pheromone receptor allowing it to
interact with a pheromone encoded in the same B mat-
ing type locus or altered the specificity of a pheromone
permitting it to recognize the self-pheromone receptors
[26, 47, 48]. Thus, reaction to self-pheromones is lead-
ing to self-compatibility. A natural precedent for an
autocrine signalling by wild type mating type phero-
mones is established in the bipolar C. neoformans pos-
sessing haploid yeast cells of the two mating types Matα
and Mata, the first of which naturally undergoes low
level of haploid fruiting due to a background self-
reaction by its pheromones. Deletion of mating type
pheromone genes causes a defect in homothallic fruit-

Journal of Basic Microbiology 2010, 50, 442 – 451

Mating-type-like genes in Moniliophthora perniciosa 449

www.jbm-journal.com

ing and overexpressing of mating type pheromones in
Matα cells enhances the frequency of the fruiting [49,
50]. Higher pheromone concentrations may thus over-
come poor binding to existent but badly fitting phero-
mone receptors.
  In some basidiomycetes, bipolarity was obviously
achieved by loss of the mating type control function of
B-mating-type-like loci. The bipolar Agaricomycete Co-
prinellus  disseminatus has such a locus with pheromone
and pheromone receptor genes functional in regulation
of dikaryon maintenance and fruiting but it is not
linked to the single mating type locus with the HD1 and
HD2 genes and it has no role in mating [10]. Phyloge-
netically, the C. disseminatus pheromone receptors clus-
ter closely with the B mating type pheromone receptors
of  C. cinerea and L. bicolor [13] as two of the seven
M. perniciosa pheromone receptors described in this
study (Fig. 2). Moreover, in addition to B mating type or
B-mating-type-like genes, tetra- and bipolar Agaricomy-
cetes can have more distantly-related non-mating-type
pheromone receptor genes as well as non-mating-type
pheromone genes [9–11, 13, 17, 25]. In C. neoformans,
next to the two mating-type-specific pheromone recep-
tors CnSTE3a and CnSTE3α, a non-mating-type phero-
mone receptor homolog (CnCpr2 in Fig. 2) has recently
been described that, strikingly, acts constitutively. Ex-
pression levels determine whether CnCpr2 can overtake
regulatory functions exerted normally in mating and
sexual reproduction by pheromones and pheromone
receptors of opposite mating type and in haploid fruit-
ing in Matα cells by an autocrine mating-type phero-
mone signalling loop [20]. It is hence possible that any
of the potential non-mating-type pheromone receptors
discovered in the various Agaricomycetes including
here the homothallic M. perniciosa C-biotype may simi-
larly act constitutively or, alternatively, in response to
pheromones encoded in mating type loci or elsewhere
in the genomes.
  There are more possibilities to overcome the need for
compatible mating in sexual reproduction, as loss
of function mutants of gene pcc1 in C. cinerea mimic
A-mating-type-regulated dikaryons [28, 29], overexpress-
ing  clp1  C. cinerea transformants produce clamp cells
[30] and S. commune transformants fruit upon ectopic
insertion of frt1 [31, 32]. Thus, changes in genes down-
stream to the mating type products can bypass the need
of mating. In the M. perniciosa genome, we found poten-
tial homologues for clp1 and frt1. The list of genes de-
tected in this study therefore offers several options as
how primary homothallism may have been generated
in this fungus.

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