Microbiology (2003), 149, 2373–2384
DOI 10.1099/mic.0.26371-0
An ordered collection of expressed sequences
from Cryphonectria parasitica and evidence of
genomic microsynteny with Neurospora crassa and
Magnaporthe grisea
Angus L. Dawe, Vanessa C. McMains, Maria Panglao,3 Shin Kasahara,4
Baoshan Chen1 and Donald L. Nuss
Correspondence
Donald L. Nuss
Center for Biosystems Research, University of Maryland Biotechnology Institute, 5115 Plant
Sciences Building, College Park, MD 20742, USA
[email protected]
Received 28 March 2003
Revised
12 June 2003
Accepted 13 June 2003
Cryphonectria parasitica, the causative agent of chestnut blight, has proven to be a tractable
experimental system for studying fungal pathogenesis. Moreover, the development of infectious
cDNA clones of C. parasitica hypoviruses, capable of attenuating fungal virulence, has provided the
opportunity to examine molecular aspects of fungal plant pathogenesis in the context of
biological control. In order to establish a genomic base for future studies of C. parasitica, the authors
have analysed a collection of expressed sequences. A mixed cDNA library was prepared from RNA
isolated from wild-type (virus-free) and hypovirus-infected C. parasitica strains. Plasmid
DNA was recovered from individual transformants and sequenced from the 59 end of the insert.
Contig analysis of the collected sequences revealed that they represented approximately 2200
individual ORFs. An assessment of functional diversity present in this collection was achieved by
using the BLAST software utilities and the NCBI protein database. Candidate genes were identified
with significant potential relevance to C. parasitica growth, development, pathogenesis and
vegetative incompatibility. Additional investigations of a 12?9 kbp genomic region revealed
microsynteny between C. parasitica and both Neurospora crassa and Magnaporthe grisea, two
closely related fungi. These data represent the largest collection of sequence information currently
available for C. parasitica and are now forming the basis of further studies using microarray analyses
to determine global changes in transcription that occur in response to hypovirus infection.
INTRODUCTION
Genomic studies have provided a wealth of information
for numerous organisms, including many fungi (reviewed
by Bennett & Arnold, 2001). The recent completion of the
Neurospora crassa genome (Galagan et al., 2003) has
provided the fungal research community with a vast
3Present address: Children’s National Medical Center, Center for
Genetic Medicine, 111 Michigan Avenue NW, Washington DC 20010,
USA.
4Present address: Department of Molecular Biology, Keio University
School of Medicine, 144 : 8 Ogura, Saiwai, Kawasaki, Kanagawa 2120054, Japan.
1Present address: Biotechnology Research Center, Guangxi University,
13 Xuiling Road, Nanning, Guangxi 530005, PR China.
Abbreviations: EST, expressed sequence tag; GO, gene ontology.
The GenBank accession numbers for the sequences determined in this
work are CB686454–CB690670.
Tables of clones classified according to molecular function and
biological process are available as supplementary data with the online
version of this paper at http://mic.sgmjournals.org.
0002-6371 G 2003 SGM
resource of data. This success is being supplemented
by efforts to make more widely available sequence data
prepared in the private sector for the economically important rice pathogen Magnaporthe grisea (www-genome.wi.
mit.edu/annotation/fungi/magnaporthe/) and the model
fungus Aspergillus nidulans (www-genome.wi.mit.edu/
annotation/fungi/aspergillus/index.html). As an alternative
to sequencing a genome, analyses of expressed sequence tags
(ESTs) have also provided a large quantity of gene identification information (reviewed by Skinner et al., 2001). Recent
descriptions of EST sequencing projects from phytopathogenic fungi include Phytophthora infestans (Kamoun et al.,
1999), Mycosphaerella graminicola (Keon et al., 2000),
Blumeria graminis (Thomas et al., 2001) and Magnaporthe
grisea (Kim et al., 2001). This approach has the benefit of
producing substantial quantities of data from organisms
that may not be the focus of larger-scale genome sequencing
ventures. The Internet has provided considerable utility as a
universal method to disseminate such information, with
many web sites such as COGEME (cogeme.ex.ac.uk: Soanes
et al., 2002) that provide search-and-retrieve access to
sequence information from one or several organisms.
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Printed in Great Britain
2373
A. L. Dawe and others
The focus of our studies, the chestnut blight fungus
Cryphonectria parasitica, was accidentally introduced into
the United States in the early part of the twentieth century
(Merkel, 1906) and has been responsible for the destruction of the native population of the American chestnut
throughout almost the entire natural range of this hardwood
species. C. parasitica is a Sordariomycete, a classification of
ascomycetes that includes many phytopathogenic fungi,
including the genera Ophiostoma (containing the species
responsible for Dutch elm disease) and Magnaporthe.
Further interest in C. parasitica stems from the observation
that natural populations of the fungus can contain virulenceattenuating dsRNA elements, or hypoviruses (Anagnostakis
& Day, 1979). The application of molecular biology techniques enabled the development of rigorously tested hypovirus
and C. parasitica reverse genetics systems (reviewed by
Dawe & Nuss, 2001). Subsequent studies led to the isolation
and characterization of a number of genes important for
virulence, including components of G-protein signalling
pathways (Choi et al., 1995; Kasahara & Nuss, 1997;
Kasahara et al., 2000; Parsley et al., 2003). Consistent with
the prediction that hypoviruses alter host phenotype by
modulating cellular signal transduction pathways, differential mRNA display (Chen et al., 1996) indicated that
infection with hypovirus CHV1-EP713 results in a large
change in the expression profile. While informative, differential display analyses do not readily reveal which host genes
are differentially expressed. Cloning and investigation of the
C. parasitica gene termed 13-1 has allowed the construction
of a promoter-based reporter system with which to further
analyse viral determinants that influence cellular signal
transduction pathways (Parsley et al., 2002). However, such
studies are also limited due to the use of a single-gene
readout to monitor pathway activity, which may not reflect
larger, global, changes occurring through the whole
transcriptome.
As part of our continuing efforts to understand the interactions of hypoviruses and their host, C. parasitica, and the
factors that affect fungal virulence, we have undertaken a
sequencing project to generate a collection of expressed
sequences. At the time of writing, only about 40 C. parasitica
genes were represented in the NCBI databases. In the
absence of a large-scale sequencing effort, we describe below
the identification of approximately 2200 new genes using a
moderate-throughput approach. By comparison of the
recovered sequences to the publicly available information
from NCBI, we demonstrate that these ESTs relate to a wide
variety of genes with known roles in other organisms and
thus provide for preliminary functional assignment of 1255
genes. Further, we explore a small region of genomic microsynteny that appears conserved across three genera and may
have functional relevance to G-protein signalling. We
anticipate that this large increase in available sequence
data for C. parasitica will prove of considerable utility to the
phytopathogenic research community while also enabling
future studies that will take advantage of new microarray
2374
technologies to examine large-scale transcriptional alterations under a variety of conditions.
METHODS
Fungal strains and growth conditions. Cryphonectria parasitica
strain EP155 (ATCC 38755) and strain EP155 infected with the prototypic severe hypovirus CHV1-EP713 [designated EP713 (ATCC
52571)] were used as representatives of wild-type and virus-infected
strains, respectively. Both strains were maintained on solid medium
[3?9 %, w/v, Difco potato dextrose agar (Becton Dickinson)].
Preparative cultures for RNA recovery were grown for 7 days at
room temperature under ambient light with cellophane on the agar
surface to permit mycelial harvest.
RNA extraction and library construction. Single-stranded RNA
was prepared from solid-medium-grown EP155 and virus-infected
cultures according to Chen et al. (1996). Poly(A)+ mRNA was isolated with oligo-d(T) cellulose (Gibco-BRL). Double-stranded cDNA
was synthesized and SalI adapters added, then equal amounts of the
cDNA from both EP155 and virus-infected EP155 were mixed prior
to cloning using the SuperScript Lambda system (Gibco-BRL/
Invitrogen) according to the manufacturer’s instructions. The in
vitro packaging of the resulting phage constructs was accomplished
with the Gigapack III procedure (Stratagene) and Escherichia coli
strain Y1090 (ZL) for phage propagation and subsequent storage.
Finally, the prepared phage were used to infect E. coli DH10B(Zip)
(Gibco-BRL/Invitrogen), which permitted plasmid excision resulting
in colonies containing cDNA constructs representing both mRNA
populations. Individual colonies were transferred into 96-well microtitre plates for cataloguing and storage. Preliminary insert size verification was performed on 20 randomly selected colonies by miniprep
and restriction digestion with NotI/SalI. For subsequent experiments
not included in this paper, all the clones that provided good
sequence data were PCR amplified using T7 and SP6 primers,
permitting a larger sampling of insert size data. An additional 120
randomly selected clones were sized in this manner.
cDNA sequence analyses. Plasmids were prepared from cultures
of stored colonies using the Qiaprep 8 system (Qiagen) with a
vacuum manifold. Each preparation was checked for yield by electrophoresis, and then submitted to the DNA Sequencing Core
Facility at the Center for Biosystems Research for analysis on ABI
3100 or 377 (Applied Biosystems) machines. Single sequence reads
in one direction from the 59 end of the cloned fragment were
obtained using the M13 reverse primer. Results files were scanned
using the SeqMan unit of DNAStar running on a Macintosh G4
computer for the presence of known vector sequence and quality of
data so that erroneous results (such as those plasmids with no
insert, or multiple templates per reaction) could be removed. Short
sequences (less than 100 bp) were also eliminated at this time.
Following cleanup, the sequences were assembled into contigs using
SeqMan and minimum match percentage of 80 % across at least
50 bp. The resulting contigs combined the similar sequences of
duplicate clones or different clones of the same original cDNA product, thus providing an approximation of the number of individual
sequences represented in the dataset. Analysis of the total sequence
data was then accomplished in two stages, both performed on an
IBM PC running the Red Hat Linux operating system and components of the BLAST program suite (Altschul et al., 1997). In the
first stage, a library of DNA sequences was prepared from the
known C. parasitica items in the NCBI database combined with
unpublished sequences from our laboratory. These sequences were
then compared, using the BLASTN algorithm, to all of the unknown
individual EST sequences (not contigs) so that clones representing
known C. parasitica genes could be identified. The second stage used
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Microbiology 149
EST sequences from C. parasitica
the EST sequences in a BLASTX analysis against the entire nonredundant protein database as available from NCBI. Tables that
comprised lists of the clones and their matches, together with NCBI
information and alignments for each hit, were generated from the
BLAST output using BioPerl (http://www.bioperl.org) software
tools. Gene ontology (GO) information was manually added using
the Gene Ontology Consortium’s Amigo browser (http://www.
godatabase.org/cgi-bin/go.cgi). The entire collection of redundant
sequences has been submitted to the NCBI (accession numbers for
dbEST, 17474474–17478690, corresponding to GenBank numbers
CB686454–CB690670) and are also publicly searchable at the COGEME
phytopathogen database (cogeme.ex.ac.uk).
Genomic sequence analyses. An EcoRI–EcoRI fragment approximately 15 kb in length isolated during characterization of the
C. parasitica G-protein b-subunit gene (cpgb-1; Kasahara & Nuss,
1997) was subcloned into pBluescript II SK+ (Stratagene). Following
restriction mapping, this insert was divided into smaller sections
and subcloned into pBluescript II SK+ or pUC18 to facilitate
sequencing, with specific primers being designed to read sequence
where required. Sequencing was performed at the CBR core facility
and at Keio University, Tokyo, Japan. Assembly of the sequence
information with reads in both directions was accomplished using
the SeqMan utility of DNAStar. The single contig obtained was
determined to accurately represent this region of the C. parasitica
genome by comparing the location of the restriction sites predicted
with those determined experimentally above. All genomic information for Neurospora crassa and Magnaporthe grisea was obtained
from the publicly available databases at the Whitehead Institute/MIT
Center for Genome Research (www-genome.wi.mit.edu). Data for
Saccharomyces cerevisiae and Schizosaccharomyces pombe were obtained
from publicly available information at the Saccharomyces genome
resource (genome-www.stanford.edu/Saccharomyces/) and the Sanger
Institute (http://www.sanger.ac.uk/Projects/S_pombe/), respectively.
RESULTS AND DISCUSSION
Sequence analyses
Given the lack of sequence data available for C. parasitica, we
embarked upon the creation of a library to permit largerscale transcriptional profiling in future studies. A library
designed to reflect the genes expressed in the presence or
absence of hypovirus was prepared by mixing cDNA derived
from two sources of mRNA: wild-type C. parasitica strain
EP155 and EP155 infected with the prototypic virulenceattenuating hypovirus, CHV1-EP713 (strain EP713). This
approach was taken to ensure representation within the
library of genes whose expression was greatly reduced in
either circumstance. Repeated rounds of infection with the
phage library and colony picking were performed to
generate between 10 and 20 new microtitre plates on each
occasion until 60 plates were completed, representing
5760 cDNA clones. Thus catalogued by plate number and
coordinate, plasmid minipreps were performed on each,
except for plates 38–44, which generated consistently poor
yields and were discarded. Six hundred and thirty-four
additional colony losses were accounted for by poor growth,
insufficient plasmid yield or handling error. By analysing
PCR products or restriction-digested plasmid preparations
from a random selection of 140 constructs, 95?7 % of the
inserts were found to be larger than 500 bp, with the vast
majority being in the 500–2000 bp range (data not shown).
All sequence data were generated by single-pass reads of the
insert from the 59 end. In one case (53F04) a chimeric insert
was found to contain two unrelated cDNA sequences
(cryparin and a sequence highly similar to kinesin-related
protein KIF1C from N. crassa), but no other cloning
anomalies were found. The mean read length after vector
removal and quality analysis was 608 bp, often including
poly(A) regions at the 39 end. Sequence files that were not
included in the following analyses because they were
determined to be of insufficient quality, or did not contain
an insert of at least 100 bp, totalled 231. Following vector
and quality trimming, a total of 2 703 587 bp of DNA
sequence were included, representing data derived from
4216 clones. Assembly of this sequence information into
contigs permitted an estimation of the number of individual
sequences included. Totalling the number of contigs present
under the particular match parameters as described (see
Methods) indicated that 2200 individual gene transcripts
were represented but, since the number of contigs obtained
in this manner can fluctuate according to the exact matching
parameters applied and there is the possibility that two
clones may represent different portions of the same coding
sequence without overlapping, this number can only be
regarded as an approximate value. More than 1600 of these
were present only once (unisequences), while the multiplyrepresented clones followed the distribution shown in
Fig. 1. (a) Distribution of the number of
clones per contig following assembly. Omitted
for clarity are the contigs corresponding to
the two highly abundant sequences (see
text) that contain more than 270 clones
each. (b) Distribution of the individual hit E
values returned from the BLASTX analysis.
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2375
A. L. Dawe and others
Fig. 1(a), with only 28 contigs containing more than 10
clones each. Two of these contigs represented highly
abundant mRNA species described below.
Galagan et al. (2003) reported that the genome size of
N. crassa was approximately 40 Mb, similar to the ~45 Mb
predicted for C. parasitica (Dr Bradley Hillman, Rutgers
University, personal communication). Given the close
phylogenetic relationship of these two fungi, it is reasonable
to postulate that the number of genes in C. parasitica is
comparable to the 10 082 currently predicted for N. crassa.
Therefore, in terms of the genome, the collection described
here probably represents between 20 and 25 % of the total
gene coding potential of C. parasitica.
Identification of known C. parasitica sequences
A BLASTN analysis was performed using a specific collection
of DNA sequences consisting of the publicly available
C. parasitica information, as well as unpublished data from
the Nuss laboratory, in comparison to our entire EST
sequence data. In this manner we determined that 598
clones represented 13 known C. parasitica genes as detailed
in Table 1, 12 from NCBI submitted data and one from
unpublished information. Two cDNA sequences were present
in far greater abundance than any others and represented
almost 6 % of the total sequences each. These corresponded
to ORF B of hypovirus CHV1-EP713 and cryparin, a
secreted hydrophobin that has previously been shown to be
highly expressed (Zhang et al., 1994). Of the 40 sequences
from NCBI that were included in this analysis, the fact that
12 were contained within our collection correlates with the
projection of approximately 25 % coverage above.
Assignment of putative function by
BLASTX
Putative identification of all of the unknown clones was
achieved using the BLASTX software tool after removal of the
known C. parasitica sequences. The remaining 3618 individual EST sequences were used for this analysis instead of
contigs to avoid any problems created by artifacts from the
contig assembly, although this approach lengthened processing time considerably. Once completed, the data were
sorted using the expect (E) value as a measure of significance
of the hit, with E>161022 classed as insignificant. This
yielded 3009 clones with hits and 609 with no significant hit.
This list was then trimmed manually to remove multiple
representatives of the same hit. Thus, 1734 individual hits
were recovered distributed across a range of E values as
described in Fig. 1(b). To further sort the data and provide
some relevant functional information, this list was truncated
to include only those 1255 examples whose hits returned an
E value of 1610210 or less. Ontology information was then
added manually to create two tables of potential groupings
relating to both molecular function and biological process
according to the definitions outlined by the Gene Ontology
Consortium (www.geneontology.org). Both entire lists are
available as electronic supplementary data with the online
version of this article (mic.sgmjournals.org). It should be
noted, however, that this provides only an indication of
functional activity or relevance to a particular process, but
does not reflect fully the non-redundant and dynamic
nature of the GO database terms. These GO terms were
developed such that one protein may well fit into several
categories (Ashburner et al., 2000), thus necessitating a
subjective assessment of the most appropriate single group
for assignment in our studies. To examine the groups of
sequences isolated, the complete dataset has been used to
generate pie charts that illustrate the distribution of the hits
obtained across the various molecular function and
biological process (Fig. 2) categories. In both cases, a significant number (almost 30 %) of the hits related strongly to
known genes of unknown function. In the case of molecular
function, enzymes constituted a further 40?9 %, of which the
hydrolases (27?6 %) and oxidoreductases (28?2 %) were
most prevalent (Fig. 2a). Of the structural proteins identified, the majority (almost 80 %) were ribosomal subunits,
with cytoskeleton and cell wall structural components
Table 1. Known C. parasitica sequences identified in the CEST collection by
Gene name
ORF B
crp-1
eapC
epn-1
gpd-1
cpc-1
7-8
cpg-2
cpk-1
eapB
cpg-1
lac-1
Small subunit
2376
BLASTN
Description
No. of sequences
NCBI gi no.
Reference
Hypovirus CHV1-EP713
Hydrophobin
Acid proteinase
Endothiapepsin
Glyceraldehyde-3-phosphate dehydrogenase
Cross-pathway control protein
Uncharacterized
Ga subunit
MAP kinase kinase
Acid proteinase
Ga subunit
Laccase
rRNA gene
277
270
17
9
8
6
2
2
2
2
1
1
1
9626820
1706154
2133287
416748
120690
4033378
Not submitted
563253
4106374
2133286
563251
250714
13540017
Shapira et al. (1991)
Zhang et al. (1994)
Jara et al. (1996)
Razanamparany et al. (1992)
Choi & Nuss (1990)
Wang et al. (1998)
Nuss lab. (unpublished)
Choi et al. (1995)
Unpublished
Jara et al. (1996)
Choi et al. (1995)
Choi et al. (1992)
Zhang & Blackwell (2001)
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Microbiology 149
EST sequences from C. parasitica
Fig. 2. Pie chart representations of the significant hits returned by BLASTX analysis classified according to (a) Molecular
Function, expanded to show further division of the Structural and Enzyme categories; and (b) Biological Process, expanded to
show divisions within the Cell Growth and Maintenance category.
making up a further 19 %. In terms of the biological
processes with which these proteins are associated, more
than two-thirds fell into the category of cell growth and
maintenance, with far fewer grouped in cell communication
(3?1 %) or development (0?8 %; Fig. 2b). Three-quarters of
those sequences classified in the cell growth and maintenance cluster were involved in metabolic processes. From
the combined observations in Fig. 2, we conclude that the
C. parasitica sequences identified are broadly representative
of the entire population of expressed genes in this organism.
Identification of genes relevant to growth,
development and pathogenicity of C. parasitica
Specific details of the following clones that have returned
hits of potential interest are noted in Table 2. This table does
not represent an exhaustive list of proteins from any functional category; for that information the reader is referred to
the electronic supplementary data (mic.sgmjournals.org).
The interaction between a pathogen and its host is dynamic,
requiring the pathogen to continually adjust to changes in
the microenvironment and to resistance factors produced by
the host. Stress response proteins may fulfil roles to maintain
homeostasis under these constantly variable conditions,
http://mic.sgmjournals.org
thus contributing to virulence. This may be manifested in
the adjustment of polyol accumulation to cope with
hyperosmotic stress as is the case for Cladosporium fulvum
(Clark et al., 2003) and M. grisea (Dixon et al., 1999) or
production of superoxide dismutases by the human pathogen
Candida albicans to protect against oxidative stress (Hwang
et al., 2002). Stress-response proteins account for 1 % of the
hits classified as cell growth and maintenance (Fig. 2) and
include a halotolerance protein from Sch. pombe involved in
inositol metabolism, as well as superoxide dismutases from
Claviceps purpurea, Glomerella cingulata, both plant pathogens, and A. nidulans. Furthermore, we noted the presence
of genes corresponding to a general stress-response protein
STI35 identified in several species of Fusarium (Choi
et al., 1990), and a protein termed GRG-1 from Podospora
anserina that is specifically enhanced under conditions of
carbon starvation (Kimpel & Osiewacz, 1999). These
sequences may represent genes involved in mechanisms of
resistance and/or response to host factors that influence
phytopathogenicity of C. parasitica.
Given the dynamic host–pathogen interface, the importance
of pathways that transmit signals from the cell surface of
the pathogen to the nucleus cannot be overstated. Our
laboratory has previously identified a number of signal
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CEST clone*
BLASTX
analysis described in the text
Name of match*
E value*
NCBI gi no.*
unnamed
3610240
19075730
Sch. pombe
14E02, 05D06, 27C05, 35B03, 54B01, 59C05,
11G08, 56F11, 27G10, 55C10, 13C07, 01B09
28A03
SOD1
1610274
15139865
STI35
1610243
135112
C. purpurea
and others
F. oxysporum
03E10, 14F04, 16C07, 18E03,
27C11, 28C01, 46F11, 47E12
Cell signalling/growth regulation
11H07, 18H09, 26F07, 50E10
GRG-1
6610215
5051406
RHO1
3610284
15293426
51B05
01D03
32H06
Rho2p
RHO3
Drab11
3610234
2610270
4610260
06C10
24A04
RIP3
ALEA
36D07
Structural proteins
17E11, 60A05, 21C02, 32H02, 33D09, 58F08
Stress response
03D12
Organism*
Description/function
Reference
Microbiology 149
Putative halotolerance protein, inositol
metabolism
Superoxide dismutase
Wood et al. (2002)
(Genome sequence)
Unpublished
Thiazole biosynthesis, stress-inducible
protein
Induced upon carbon starvation
Choi et al. (1990)
Kimpel & Osiewacz (1999)
Rho family small GTPase
Unpublished
19115481
13432036
18376163
B. graminis
and others
Sch. pombe
A. fumigatus
N. crassa
Rho family small GTPase
Rho family small GTPase
Probable GTP binding protein
3610212
5610232
5453419
18376134
D. discoideum
N. crassa
Ras-interacting protein
Related to aimless RasGEF
Sbp1p
1610245
19111914
Sch. pombe
Affects mitosis-interphase transition
Nakano & Mabuchi (1995)
Beauvais et al. (2001)
Galagan et al. (2003)
(Genome sequence)
Lee et al. (1999)
Galagan et al. (2003)
(Genome sequence)
He et al. (1998)
TUB-1
16102104
12963224
a-Tubulin
Takano et al. (2001)
45E09, 46C02
26F08, 05B02, 57H08, 50H03, 60C01
31B07, 51B07
TBG
ActA
DFG5
5610241
16102121
8610294
1729857
14194449
16944622
C. lagenarium
and others
N. crassa
A. chrysogenum
N. crassa
35A02
04E11
12C01
ASPB
PCD1
unnamed
2610296
16102111
2610211
1791305
5725417
19114968
A. nidulans
P. brassicae
Sch. pombe
13B10
Cct7p
2610257
6322350
S. cerevisiae
60G04
Sop2p
1610251
11359003
Sch. pombe
Light detection/response
37B08
VVD
1610238
12831203
N. crassa
47H02, 28C02
CCG6
4610223
8488971
N. crassa
P. anserina
c-Tubulin
c-Actin
Heckmann et al. (1997)
Diez et al. (2000)
Cell wall biogenesis in S. cerevisiae
Galagan et al. (2003)
(Genome sequence)
Septin B
Momany & Hamer (1997)
Putative septin
Singh et al. (2000)
Similar to Dam1p required for spindle
Wood et al. (2002)
maintenance in S. cerevisiae
(Genome sequence)
Chaperonin component, affects cytoskeleton Rasmussen (1995)
organization/biogenesis
Arp2/3 complex subunit
Balasubramanian et al.
(1996)
Clock-controlled repressor of light
regulated processes
Clock-controlled protein
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Heintzen et al. (2001)
Bell-Pedersen et al. (1996)
A. L. Dawe and others
2378
Table 2. Details of the putative protein identifications based on
http://mic.sgmjournals.org
*Where multiple clones matched the same entry repeatedly, or the same protein hit from different organisms, only the best match (lowest E value) is given with corresponding name, gi number
and organism of origin. Subsequent CEST clones are listed in match order (increasing E value).
Mattjus et al. (2003)
Espagne et al. (2002)
Barreau et al. (1998)
Loubradou et al. (1997)
Smith et al. (2000)
P. anserina
P. anserina
P. anserina
P. anserina
N. crassa
7610247
2610244
5610216
16102103
3610215
HET-C
HET-D
MOD-A
MOD-E
HET-6
2133323
17225210
7452504
6016265
6578941
Glycolipid transfer
b-Transducin-like WD-40 protein
Proline-rich SH-3 domain protein
HSP90 homologue
TOL/HET-6/HET-E domain, linked to
other incompatibility genes
Bieszke et al. (1999)
N. crassa
5565892
1610
19F01
Vegetative incompatibility
12A07, 55F11, 12E11
19B12
13B11, 12D10
60H02, 06E05, 25F07
21B02, 18G09, 12G12
255
NOP-1
Organism*
CEST clone*
Table 2. cont.
Name of match*
E value*
NCBI gi no.*
Opsin
Description/function
Reference
EST sequences from C. parasitica
transduction components, including the G-proteins CPG-1,
CPG-2 (Choi et al., 1995), CPG-3 (Parsley et al., 2003) and
CPGB-1 (Kasahara & Nuss, 1997) as well as other proteins
required for their function such as BDM-1 (Kasahara et al.,
2000) and a regulator of Ga, RGS (G. C. Segers & D. L. Nuss,
unpublished). Identifying specific sequences of interest in
our collection, we have also noted a number of other genes
that appear to be involved in perception and transfer of
signals. These include additional internal transducers: examples of the small GTPase RHO family, a RAB family member,
Drab11, and a RAN-binding protein (Sbp1p) probably
involved in cell cycle control. Other genes of predicted
importance for growth include DFG5, a glycosylphosphatidylinositol (GPI)-anchored protein essential for cell
wall biogenesis in S. cerevisiae (Kitagaki et al., 2002) and
two septins. Similarly, cellular structural components were
found (making up 11?6 % of the structural protein category,
Fig. 2a), including tubulin and actin as well as components
that regulate their organization and biogenesis such as
Dam1p, Cct7p and the Arp2/3 complex. Chen et al. (1996)
noted the alteration of cAMP levels in response to hypovirus
infection; therefore the identification of a RAS-interacting
protein (RIP3) that, in Dictyostelium discoideum, is required
for chemotaxis and cAMP-mediated signal transduction
(Lee et al., 1999) is potentially relevant. Most interestingly,
we have also identified an orthologue of the Ras guanine
nucleotide exchange factor (RasGEF) AleA, a likely functional partner of RIP3. Together, these two components may
form part of a C. parasitica RAS-regulated pathway as has
been postulated for D. discoideum (Lee et al., 1999).
During growth of C. parasitica on solid medium, rings of
developmentally distinct regions become apparent, areas
that are primarily the sites of asexual sporulation and
pigmentation. The frequency of the occurrence of these
bands corresponds to the light cycle in which the culture is
being grown and thus provides evidence of the lightresponsive nature of these processes. In N. crassa, substantial
advances have been made in studying the mechanism of
light response and maintenance of circadian rhythms upon
which similar developmental steps including conidiation are
dependent (reviewed by Dunlap, 1996). One key component
of the process is the PAS protein VVD (Heintzen et al.,
2001). We have identified an orthologue of this gene as well
as a clock-controlled gene whose expression in N. crassa is
dependent on the clock cycle (Bell-Pedersen et al., 1996) and
a cDNA similar to the nop1 gene of N. crassa that encodes a
putative rhodopsin orthologue (Bieszke et al., 1999). In an
additional effort unrelated to the C. parasitica EST library,
we have also recovered a partial sequence of the transcriptional regulator FRQ from C. parasitica by degenerate PCR
(A. L. Dawe & D. L. Nuss, unpublished observations).
Further study of the mechanisms of light response and
circadian rhythm maintenance may provide insights into
the importance of these developmental regulatory pathways
for fungal plant pathogenicity.
Our studies are also predicated in part upon efforts to
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A. L. Dawe and others
enhance the naturally occurring virulence attenuation
characteristics of the virus family Hypoviridae as a method
of biological control (reviewed by Dawe & Nuss, 2001).
Effective dissemination of the hypovirus through a native
population requires the passing of the dsRNA elements from
infected to non-infected fungus by means of anastomosis
(hyphal fusion: Anagnostakis & Day, 1979). This process is
dependent upon the vegetative incompatibility (vic) loci of
each strain, and virus transmission can be greatly inhibited
by heteroallelism between pairs (Cortesi et al., 2001). At the
time of writing, public information concerning the sequence
of the vic genes was unavailable, but we have isolated five
new C. parasitica sequences in this study that appear to be
related to this important process. Genetic analysis of the
fungus P. anserina has identified at least 10 loci (het-c, -d and
-e; mod-a, -d and -e; idi-1, -2 and -3; pspA), as being involved
in the vegetative incompatibility reaction (reviewed by Glass
& Kaneko, 2003) and their characterization has revealed
proteins probably involved in signal transduction and
membrane structural modifications. The HET-C protein has
recently been shown to increase glycosphingolipid transfer
rates (Mattjus et al., 2003) and also is involved in growth and
sporulation (Saupe et al., 1994). Our analysis yielded three
EST clones corresponding to het-c. Complete sequencing of
these inserts from both 59 and 39 ends has enabled us to
construct a complete predicted amino acid sequence of the
C. parasitica HET-C orthologue. When compared to the
sequence for het-c of P. anserina and the predicted protein of
N. crassa locus NCU07947.1, significant identity throughout
the polypeptides is clear (Fig. 3). Each protein is 53–63 %
identical to the other two, suggesting considerable conservation of incompatibility components between these
fungi. This is further supported by the presence of
C. parasitica ESTs corresponding to the vegetative incompatibility proteins HET-D, a WD-40 domain protein of
unknown function from P. anserina (Espagne et al., 2002)
and HET-6 of N. crassa, whose function is also presently
unspecified. The mod genes of P. anserina are believed to
code for signalling components that mediate the incompatibility response that can lead to programmed cell death.
MOD-A and MOD-E were represented in the C. parasitica
library and a sixth C. parasitica gene, the Ga subunit CPG-2
identified by Choi et al. (1995), may also be relevant since it
is closely related to the MOD-D protein (Loubradou et al.,
1999). In contrast to the CPG-1 a subunit, which caused
extensive phenotypic changes when absent, deletion of cpg-2
caused only minor changes from wild-type and therefore
was not required for pigmentation, sporulation or virulence
(Choi et al., 1995). Further analysis of the clones identified
in this study may, in conjunction with characterized loci in
C. parasitica and other organisms, provide additional
insights into this phenomenon and its relevance to the
engineering of hypoviruses for enhanced potential as
biological control agents.
A representation of the genera of the organisms from which
the best BLASTX results for the EST clones were derived is
given in Fig. 4. To avoid skewing of the data due to the
presence of large numbers of hits from highly analysed
genomes, we have scored each genus only once. Thus, the pie
charts depict a breakdown of the 132 individual genera
represented in the hits analysed above. As expected, most of
the highly homologous hits are obtained with sequences
from ascomycetous fungi. Narrowing the list to follow the
taxonomy of C. parasitica, it is evident that most of the
hits are from phylogenetically related organisms. The final
grouping represents the Sordariomycetes and contains
many phytopathogenic fungi including the genera Claviceps,
Glomerella, Gibberella, Nectria, Ophiostoma, Rosellinia
and Magnaporthe. N. crassa, while not a pathogen, is also
a member of this group. Of the 33 genera from the
Pezizomycotina group of ascomycetes that yielded BLASTX
hits of E<1610210, 16 are plant pathogens and a further six
are pathogens of other organisms. Continued study of the
sequence data presented here may well illuminate specific
pathways and genes that are common between pathogenic
fungi and required for virulence.
Genomic microsynteny among Sordariomycetes
During investigations in our laboratory of signal transduction pathways that modulate virulence in C. parasitica, we
have used the publicly available information to compare
C. parasitica genes with those present in the N. crassa
genome. Our previous studies had identified CPGB-1, a
G-protein b-subunit (Kasahara & Nuss, 1997), and a novel
phosducin-like protein called BDM-1 (Kasahara et al.,
2000). While the exact roles of these two proteins have not
been determined, it is clear from studies in C. parasitica that
BDM-1 is essential for correct CPGB-1-mediated signalling
(Kasahara et al., 2000), most likely through a direct interaction of the two proteins (A. L. Dawe & D. L. Nuss,
unpublished observations), and that the pathway(s) affected
subsequently modulate a wide variety of cellular processes
including growth, pigmentation, sporulation and virulence.
Fig. 3. PILEUP comparison of HET-C protein
sequences from P. anserina (P.a. HET-C; gi
number 537934), N. crassa (NCU07947.1)
and C. parasitica (C.p. HET-C; derived from
three completely sequenced cDNA inserts).
Only those amino acids conserved between
all three proteins are highlighted, with dark
blocks representing identical residues and
grey blocks similar residues.
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Microbiology 149
EST sequences from C. parasitica
NCU00441.1, that appeared similar to bdm-1. Furthermore,
this locus was situated in close proximity to gnb-1 (spanning
a region of approximately 7 kb) and the ORFs were oriented
in the same direction (Fig. 5a). Returning to the original l
phage clone from which the genomic sequence of cpgb-1 was
obtained, we were able to establish that part of the ORF of
bdm-1 was present at the opposite end to cpgb-1 (data not
shown). Subsequent shotgun sequencing of this entire l
clone was then correlated with existing genomic data of the
two genes and their immediate surroundings. A single
contig of 12982 bp comprising 51 sequences (23 for the top
strand, 28 for the bottom, resulting in 2?5-fold average
coverage over the entire length) was found to contain the
two genes oriented in the same manner as N. crassa
(Fig. 5b).
Fig. 4. A pie chart representation of the distribution of significant hits returned by BLASTX analysis, classified according to
the taxonomy of the organism from which the hit was derived.
Each different genus was only scored once to avoid bias from
large sequencing projects.
Similar developmental importance has been ascribed to the
gnb-1 gene of N. crassa (Yang et al., 2002). Examination
of the N. crassa genomic data revealed a gene, locus
Regions of microsynteny have been previously noted
between M. grisea and N. crassa, wherein orientations of
ORFs and relative locations were maintained (Hamer et al.,
2001). Therefore, further comparison was also made with
available data for M. grisea, although the genomic information from release 2.1 did not represent a complete
genome assembly (Whitehead Institute/MIT Center for
Genome Research, January 2003). However, the orthologues
of the Gb subunit (locus MG05201.1; also known as Mgb1,
NCBI gi number 21624377) and bdm-1 (locus MG05200.1)
were found to be located on adjacent (linked) cosmids
(Fig. 5c). The distance between the two genes cannot be
accurately determined until the intervening sequence is
released, but the relative orientation of the two reading
frames appeared to be identical to that of C. parasitica and
N. crassa. Using the COGEME phytopathogen EST database
(cogeme.ex.ac.uk/) we have also noted the presence of EST
sequences corresponding to orthologues of Gb (clone Bccon2700) and BDM-1 (clone Bc-con1300) from the plant
pathogen Botryotinia fuckeliana. Presently, appropriate
genome information is not available but it would be our
prediction that the arrangement of the BDM-1 and Gb genes
is maintained in this organism also. While published data on
the bdm-1 orthologues are not available for either M. grisea
or N. crassa, it seems likely that the protein products of these
genes will prove important to the correct function of the Gb
subunit given that this consistent arrangement of the two
genes is maintained so rigorously.
Fig. 5. A region of microsynteny between
three Sordariomycetes: conserved genomic
arrangement of two genes that encode a
G-protein b-subunit and a phosducin-like
potential regulator of Gb signalling in (a)
N. crassa, (b) C. parasitica and (c) M. grisea.
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A. L. Dawe and others
The synteny appears absent in the genomes of S. cerevisiae
and Sch. pombe, since the two genes of interest are at least
100 kb and 40 kb apart, respectively (data not shown).
Furthermore, of the two phosducin-like proteins Plp1p (gi
number 6320389; most similar to BDM-1) and Plp2p
(gi number 6324856) in S. cerevisiae (Flanary et al., 2000),
PLP2 is located closer to the b-subunit STE4. We have also
noted an EST clone (26C10, tentatively termed bdm-2)
from C. parasitica that appears to correspond to PLP2
(E=9610234), a gene that gave a lethal phenotype when
knocked out in S. cerevisiae (Flanary et al., 2000). Examining
the genome data available for N. crassa and M. grisea we
have determined that sequences that resemble PLP2/bdm-2
are present in both of these fungi also (NCU00617.1 and
MG02871.1, respectively) but are located on different
linkage groups and supercontigs. Therefore, while both
Plp1p and Plp2p were characterized as phosducin-like
proteins from S. cerevisiae, it appears as if the functional
relationship of the Plp2p family to Gb subunits may be
quite different from that which has been hypothesized for
BDM-1 (Kasahara et al., 2000) and Plp1p (Flanary et al., 2000).
Of additional note, several N. crassa EST clones were found
to map to a region approximately 700 bp downstream of the
Gb subunit, but comparison with the entire cDNA fragment
of gnb-1 (Yang et al., 2002) indicated that they represented
an untranslated region of this ORF (data not shown). There
are no other ESTs or predicted ORFs that map to this region
of the N. crassa genome. Similarly, we have not isolated any
C. parasitica ESTs that correspond to the equivalent region
in this organism. Therefore, it appears as if the two genes are
maintained in this arrangement across genera without any
intervening genes. Additional comparison of the intervening
regions of N. crassa and C. parasitica failed to indicate any
conserved regions of sequence identity or similarity (data
not shown). This suggests that the physical proximity of the
two genes is the most important feature, perhaps reflecting a
requirement for co-segregation of these loci.
The authors would like to thank Steve Screen for assistance with setting
up the BLAST utilities, Todd Allen for critical evaluation of the
manuscript, and Ellen Rosenbloom and Sharmili Mathur for technical
assistance. This work was supported in part by Public Health Service
grant number GM55981 to D. L. N.
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