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The Dawn of Fungal
Pathogen Genomics
Jin-Rong Xu,1 You-Liang Peng,2 Martin B.
Dickman,3 and Amir Sharon4
1
Department of Botany and Plant Pathology, Purdue University, West Lafayette,
Indiana 47907; email: jinrong@purdue.edu
2
Department of Plant Pathology, China Agricultural University, Beijing 100094, P. R.
China; email: pengyl@public3.bta.net.cn
3
Institute for Plant Genomics and Biotechnology, Texas A&M University, College
Station, Texas 77843; email: mbdickman@tamu.edu
4
Department of Plant Sciences, Tel Aviv University, Tel Aviv 69978, Israel;
email: amirsh tauex.tau.ac.il
Annu. Rev. Phytopathol.
2006. 44:337–66
First published online as a
Review in Advance on
April 20, 2006
The Annual Review of
Phytopathology is online at
phyto.annualreviews.org
doi: 10.1146/
annurev.phyto.44.070505.143412
c 2006 by
Copyright Annual Reviews. All rights
reserved
0066-4286/06/09080337$20.00
Key Words
fungal pathogens, pathogen genomics, fungal pathogenesis,
Magnaporthe, Fusarium, Sclerotinia
Abstract
Recent advances in sequencing technologies have led to a remarkable increase in the number of sequenced fungal genomes. Several
important plant pathogenic fungi are among those that have been
sequenced or are being sequenced. Additional fungal pathogens are
likely to be sequenced in the near future. Analysis of the available
genomes has provided useful information about genes that may be
important for plant infection and colonization. Genome features,
such as repetitive sequences, telomeres, conserved syntenic blocks,
and expansion of pathogenicity-related genes, are discussed in detail with Magnaporthe oryzae (M. grisea) and Fusarium graminearum
as examples. Functional and comparative genomic studies in plant
pathogenic fungi, although still in the early stages and limited to a
few pathogens, have enormous potential to improve our understanding of the molecular mechanisms involved in host-pathogen interactions. Development of advanced genomics tools and infrastructure is
critical for efficient utilization of the vast wealth of available genome
sequence information and will form a solid foundation for systems
biology studies of plant pathogenic fungi.
337
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INTRODUCTION
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Fungi have an enormous impact on human
welfare by destroying valuable crops as devastating pathogens or producers of mycotoxins.
Better understanding of fungal-plant interactions and pathogenicity factors is a crucial
prerequisite for the development of novel
disease control strategies. Recent advances
in sequencing and genomic techniques have
made it possible to monitor gene expression changes at the whole-genome level,
which has impacted all aspects of biological
sciences. Since the sequencing of Saccharomyces cerevisiae, members of the American
Phytopathological Society (APS) and fungal
genetics community have been actively
pursuing genome sequencing and genomic
studies. The Fungal Genome Initiative (FGI)
was initiated in 2000 to promote sequencing
of representative species across the Kingdom
Fungi that are important to medicine, agriculture, and industry (http://www.broad.mit.
edu/annotation/fgi). In 2002, APS released a
whitepaper on pathogen genome sequencing,
which has been updated frequently for the
list of pathogens recommended for sequencing
(http://www.apsnet.org/members/
ppb). In the past few years, over 40 complete fungal genomes have been publicly
released, and a similar number of fungi
are currently being sequenced (31). Several important plant pathogenic fungi are
among those that have been sequenced
or are being sequenced (Figure 1). The
NSF/USDA Microbial Genome Sequencing
Program has been the major source of
support for sequencing phytopathogenic
fungi.
Because our knowledge of molecular
mechanisms of fungal pathogenesis is limited,
comparative and functional genomic studies
offer great promise to improve our understanding of host-pathogen interactions. Several reviews have been published recently on
genomic studies in yeast, human pathogens,
and other fungi (27, 31, 107). In this review, we focus on plant pathogenic fungi and
338
Xu et al.
present an overview of genome sequencing
projects, describe features learned from sequenced genomes, and discuss our perspectives on genomic studies of fungal pathogenesis. Since Magnaporthe oryzae (formerly M.
grisea) is the only plant pathogenic fungus to
have its genome sequence published (20) and
many pathogens are being sequenced, we realize that it is exciting but challenging to prepare
this review. Most of our discussions related
to pathogenesis will be focused on Fusarium
graminearum and M. oryzae, the first two plant
pathogenic fungi sequenced in the public
sector.
GENOME SEQUENCING AND
GENOMES
Two major genome sequencing centers,
the Broad Institute (formerly Whitehead
Institute-Center for Genome Research) at
MIT and the DOE Joint Genome Institute
(JGI), have played a key role in sequencing plant pathogenic fungi. A few fungal
pathogens are being sequenced at The Institute for Genomic Research (TIGR) and
the Genome Sequencing Center at Washington University (WU-GSC). Genome sequences of several phytopathogenic fungi that
were sequenced in the private sector (at Syngenta, Bayer CropScience AG, and Exelixis)
have been publicly released through the Broad
Institute Web site (http://www.broad.mit.
edu/annotation/fgi).
Sequencing Strategies
The whole-genome shotgun (WGS) approach was used to sequence M. oryzae (20)
and most other fungal pathogens. For fungi
sequenced at the Broad Institute, random
clones from plasmid (4 kb), fosmid (40 kb),
and bacterial artificial chromosome (BAC) libraries were used. The number of end sequences generated for each fungus depended
on the genome size and available funding.
The Broad Institute also is responsible for
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Aspergillus flavus
Eurotiomycetes
Phaeosphaeria nodorum
Leptosphaeria maculans
Pyrenophora tritici-repentis
Alternaria alternata
Dothideomycetes
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Mycosphaerella graminicola
Mycosphaerella fijiensis
Sclerotinia sclerotiorum
Botrytis cinerea
Leotiomycetes
Magnaporthe oryzae
Fusarium graminearum
Fusarium verticillioides
Nectria haematococca
Fusarium oxysporum
Sordariomycetes
Ustilago maydis
Ustilaginomycetes
Puccinia graminis
Phakopsora meibomiae
Phakopsora pachyrhizi
Urediniomycetes
Figure 1
sequencing the genomes of several model filamentous fungi and human pathogens, including Neurospora crassa, Aspergillus nidulans,
and Cryptococcus neoformans, with the same
WGS strategy (29, 30). JGI has sequenced the
first saprophytic basidiomycete Phanerochaete
chrysosporium (87) and a few fungal pathogens.
Random clones were end-sequenced at JGI
from small-insert (2–4 kb) and medium-insert
(6–8 kb) plasmid libraries and one fosmid
library (35–40 kb). To date, most fungal
genome sequencing has been to 6–10X coverage (Table 1). However, for gene discovery
at private companies usually only small-insert
plasmid libraries were sequenced at lower (5X)
coverage.
Plant pathogenic
fungi that have been
sequenced (shaded )
or are being
sequenced (not
shaded ).
Genome Assembly
Advances in assembly algorithms and the inclusion of end sequences from large-insert
genomic clones in WGS have helped generate assemblies with high-sequence quality
and continuity. The Broad Institute uses the
Arachne package to assemble fungal genomes.
In brief, the two ends of the fragment in
each clone are sequenced as paired reads.
The assembly process uses the paired reads to
identify contiguous stretches of sequence as
contigs. Contigs are ordered and linked together into larger supercontigs or scaffolds
with paired reads lying in different contigs.
The genome assemblies of N. crassa, A. nidulans, and F. graminearum in most cases match
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Table 1
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Sequenced plant pathogenic fungi
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Assembled sequence
Fungal pathogen
Strain
Coverage
Contigs
Scaffolds
Length
(Mb)a
Yearb
Genes
Genic
DNA (%)
Magnaporthe oryzaec
70–15
7
739
197
39.4
2002
12,841∗
47.8
Fusarium
graminearum
PH-1
10
511
43
36.1
2003
11,640∗
56.2
Gz3639
0.4
0.4x
NAd
NA
2005
NA
NA
Stagonospora nodorum
SN15
10
496
109
37.1
2005
16,597
58.3
Aspergillus flavus
NRRL 3357
5
79
36.3
2005
13,071
Ustilago maydis
521
SB1
10
5
274
NA
48
60
19.7
19.3
2003
2004
6,522
NA
64.1
NA
Botrytis cinerea
B05–10
5.4
4534
588
38.8
2005
16,448
47.4
Sclerotinia sclerotiorum
(ATCC18683)
1980
8
679
36
38.0
2005
14,522
50.8
Nectria haematococca
(FGSC9596)
77-13-4
7
396
NA
52.4
2005
16,237
NA
Fusarium verticillioides
(NRRL 20956)
7600 (M3125)
4.2
3633
61
38.9
2005
NA
NA
a
Total length of combined contigs.
The year of initial sequence release.
c
Information of M. ozyzae is based on latest release (V2.2).
d
Information not available.
b
well with genetic maps (20, 29). F. graminearum has over 99.6% of the contigs aligned to
the genetic map, indicating that the Arachne
package works efficiently for fungal genome
assemblies. Similar methodologies are used
by the JGI’s assembler JAZZ (87). Like many
other WGS sequencing projects, all genome
assemblies of fungal pathogens have gaps.
Some of which may result from DNA sequences that are not clonable in Escherichia
coli or present difficulties for conventional
sequencing reactions. Sequencing additional
clones from different genomic libraries and
with different technologies, such as pyrosequencing (111), will be necessary to improve genome assemblies. However, all fungal
genome sequencing projects have excluded
reads that cannot be assembled. Many of these
unassembled reads are repetitive sequences
often associated with telomeres, centromeres,
and rDNA repeats. Robust automated methods are needed to include nonmitochondrial
excluded reads into genome assemblies.
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Xu et al.
Because many phytopathogenic fungi are
asexual or not well studied, genetic maps are
not available. However, at least two existing
mapping techniques are suitable for validating genome assemblies. HAPPY mapping, an
in vitro approach for determining the order
and spacing of DNA markers through PCR
assays with native genomic DNA, is independent of cloning and applicable to construction of regional or genome-wide physical maps (120). Optical mapping relies on the
ability to obtain high-molecular-weight DNA
molecules bound under tension to a derivatized glass surface. A number of restriction
enzymes and digestion schemes are used to
generate the restriction patterns of individual DNA molecules that can be visualized by
fluorescence microscopy and used to reconstruct physical maps (112). The order and
distance between restriction sites observed
in optical maps can be used for comparison
with in silico digests of genome assemblies for
validation.
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Ascomycetes
Many important plant pathogenic fungi are
ascomycetes or anamorphs of ascomycetes.
Several ascomycetous pathogens were among
the first filamentous fungi sequenced. Rice
blast, caused by M. oryzae, is one of the
most severe diseases of rice throughout the
world, and it was the first plant pathogenic
fungus with its genome sequence published
(20). Approximately 7X coverage of M. oryzae
strain 70–15 was sequenced. In addition, 38
BACs of chromosome 7, the smallest chromosome, were individually sequenced (122).
The genome assembly consists of 2273 sequence contigs longer than 2 kb, ordered and
oriented within 159 supercontigs. The total
length of all sequence contigs is 38.8 Mb (20).
Thirty-three scaffolds representing 32.8 Mb
or 85% of the draft assembly were ordered on
the genetic map, indicating that the assembly
is reasonably good despite the abundance of
repetitive sequences. The assembly also displays considerable long-range continuity. The
scaffold N50 of 1.6 Mb ( = over 50% of all
bases in scaffolds larger than 1.6 Mb) is similar to that of the N. crassa assembly (29). Recently, approximately 2X coverage each of two
M. oryzae field isolates, Y34 and P131, have
been sequenced (Y. Peng, unpublished). Preliminary analysis indicated that Y34 and P131
had additional 5.7 Mb and 1.5 Mb sequences,
respectively, that were absent in the laboratory
strain 70–15.
F. graminearum (teleomorph Gibberella
zeae) is the causal agent of Fusarium head
blight (FHB) of wheat and barley (40). The
genome assembly of approximately 10.6X sequence coverage was released to the NCBI
in 2003, making F. graminearum the second
plant pathogenic fungus with its genome sequence available to the public. The genome
assembly of stain PH-1 consists of 511 contigs larger than 2 kb. The total length of all
sequence contigs is 36.1 Mb with over 50%
of all bases residing in scaffolds larger than
5.4 Mb, which is better than all other filamentous fungi that have been sequenced. The
contigs are ordered and oriented within 43
scaffolds. The vast majority (99.8%) of the assembly has been aligned to the four F. graminearum linkage groups (33, 58). In 2005, Syngenta released its 2X genome sequence of F.
graminearum strain 3639 to the public. Both
PH-1 and 3639 are genetically similar U.S.
isolates belonging to lineage seven (101).
Syngenta also released an approximately
4X genome sequence of F. verticillioides strain
7600, a causal agent of kernel and ear rot of
maize and a producer of fumonisin mycotoxins. Additional 4X coverage of 7600 will be sequenced by the Broad Institute in 2006. The
Broad Institute also will sequence 7X coverage
of F. oxysporum. Members of the F. oxysporum
species complex cause a variety of devastating
blights, root rots, and wilt diseases. The first
fungal pathogen sequenced at JGI was Nectria haematococca MPVI, which is a member
of the F. solani complex and in the same order of Hypocreales as other sequenced Fusarium species (Figure 1). A total of 546,767
sequence reads representing 8.2X sequence
coverage have been generated and assembled
into 396 scaffolds. About half of the assembled 52.4-Mb genome sequence is contained
in scaffolds longer than 1.2 Mb (H. VanEtten,
personal communication). To date, this is the
largest ascomycete genome that has been sequenced (Table 1). The current draft release
(V1.0) includes a total of 16,237 predicted
gene models. The “conditionally dispensable”
(CD) chromosome that was first reported in
N. haematococca is known to carry habitatdefining genes (92) and may contribute to the
unusually large genome size. The genome sequence of N. haematococca will be useful to
understand the unique structural features and
evolutionary origin of the CD chromosomes.
Sclerotinia sclerotiorum and Botrytis cinerea
are two Discomycetes in the class Leotiomycetes. S. sclerotiorum is a ubiquitous
soilborne necrotrophic pathogen and causes
white mold and stem rot on a broad range
of crop plants (7). It has been well studied
for sclerotium development, pH regulation
of infection processes, and the role of oxalate
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in fungal pathogenesis. The 8X coverage sequence was assembled into 679 contigs with
a combined total of 38.0 Mb. The average
length of 36 supercontigs is 1.1 Mb with a
scaffold N50 of 1.6 Mb. An optical map is under construction for validating the genome assembly of S. sclerotiorum, which is predicted
to contain 14,522 protein-encoding genes.
The gray mold fungus B. cinerea also infects a wide range of plants. The Broad Institute has assembled WGS sequences of B.
cinerea strain B05–10 released by Syngenta in
2005. The 5.4X coverage was assembled into
4534 contigs and 588 supercontigs. The total length of combined contigs is 38.8 Mb.
Genoscope, the French National Sequencing Center, is sequencing B. cinerea strain T4
(http://www.genoscope.org). In addition to
12X coverage WGS sequences, 20,000 BAC
and 5000 full-length cDNA clones of T4 will
be sequenced as part of this project (M. Lebrun, personal communication).
The first Dothideomycete pathogen sequenced is Stagonospora nodorum (teleomorph
Phaeosphaeria nodorum). This fungus is also
known as Septoria nodorum and causes glume
blotch of wheat and other cereals. A 10X
coverage of S. nodorum was sequenced at
the Broad Institute and released to the public in 2005. The current sequence assembly has 109 supercontigs with a combined
length of 37.1 Mb. A total of 16,597 ORFs
has been predicted by automated annotation.
Another member of the Dothideomycetes
that has been sequenced is Alternaria brassicicola, to date the only plant pathogenic fungus sequenced by WU-GSC. A. brassicicola
is a necrotrophic pathogen of the Brassicaceae, including Arabidopsis. A preliminary
assembly of nearly 280,000 WGS reads of
the haploid strain ATCC 96866 revealed a
genome size of approximately 31 Mb. Approximately 10,500 genes could be identified (C. Lawrence, personal communication).
Another Dothideomycete being sequenced
at the Broad Institute is Pyrenophora triticirepentis, the causal agent of tan spot of
wheat. Genoscope is sequencing 25,000 ESTs
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Xu et al.
and a 12X genome coverage of Leptosphaeria
maculans, the causal agent of blackleg or
stem canker of canola (B. Howlett, personal
communication).
Mycosphaerella, one of the largest genera of
plant pathogenic fungi, attacks a range of temperate and tropical crops. Two Mycosphaerella
species, M. graminicola and M. fijiensis, are being sequenced by JGI. Septoria blotch caused
by M. graminicola (anamorph Septoria tritici) is one of the most important diseases of
wheat worldwide. JGI has sequenced 8X coverage of strain IPO323 (S. Goodwin, personal communication). IPO323 has an estimated genome size of 41.8 Mb and is a
parent of the mapping population that carries avirulence genes for several commonly
used wheat resistance (R) genes (68). M. fijiensis is the causal agent of the devastating
leaf streak disease of banana commonly called
Black Sigatoka. JGI will sequence 8X coverage of stain CIRAD86, which has an estimated
genome size of 40 Mb. Phylogenetic analysis
has shown that many Mycosphaerella species
originated by recent adaptive radiations on
different hosts (17). Therefore, comparative
analysis of these two species will provide useful information on genetic diversity and speciation in Mycosphaerella.
The anamorphic genus Aspergillus comprises a diverse group of fungi spanning over
200 million years of evolution, and it has
been more intensely sequenced than any other
genus of filamentous fungi. Many of these
cosmopolitan fungi are of agricultural, industrial, and medical significance, such as A.
flavus, A. oryzae, and A. fumigatus. Comparative analyses of three recently published Aspergillus genomes indicate that the 37-Mb
genome of A. oryzae contains 12,074 genes
and is 7 and 9 Mb larger than the genomes
of A. nidulans and A. fumigatus, respectively
(30, 86, 97). A. flavus is an opportunistic
plant and human pathogen that produces aflatoxin mycotoxins in infested crops. The 36.3Mb genome of A. flavus was sequenced at
TIGR and predicted to encode 13,071 genes
(http://www.aspergillusflavus.org). Other
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sequenced Aspergillus species include A. terreus, A. parasiticus, and A. fischerianus. Over
5000 noncoding regions are conserved across
all three published Aspergillus genomes and
contain potential functional elements, including a previously uncharacterized thiamin pyrophosphate (TPP) binding riboswitch (30).
In A. oryzae, syntenic blocks conserved in A.
nidulans and A. fumigatus are distributed in
a mosaic manner with A. oryzae-specific sequences, which are enriched for genes involved in secondary metabolism (86).
In addition to N. crassa and Aspergillus
species, several other nonphytopathogenic filamentous ascomycetes have been sequenced,
including Chaetomium globosum, Trichoderma
reesei, and T. virens. Trichoderma species have
been used as biological control agents for preventing fungal diseases. T. reesei, sequenced at
JGI, has seven chromosomes and a genome
size of 33 Mb. Because of its ability to secrete a
large number of extracellular lytic enzymes, T.
reesei is being developed for the production of
enzymes for the conversion of plant biomass
materials into industrially useful bioproducts.
Basidiomycetes
To date, Ustilago maydis is the only basidiomycete plant pathogen whose genome is
publicly available. It is a facultative biotrophic
pathogen that causes smut on maize and
teosinte. The intricate relationship between
mating and pathogenesis has made U. maydis
a model system for studying fungal-plant infections, particularly signal transduction pathways (61, 78). The 10X genome sequence of
strain 521 has been assembled into 48 supercontigs with a total length of 19.7 Mb. Automated annotation has identified 6522 ORFs
longer than 100 amino acid residues. Bayer
CropScience has sequenced a minimal tile
of 258 BAC clones across the 23 chromosomes and generated a 17.5-Mb assembly of
strain 521. In addition, 245,000 WHS reads
generated at Exelixis for strain FB1 (about
5X sequence coverage) were assembled into
60 supercontigs with a combined length of
19.3 Mb. Both the Bayer and Exelixis assemblies are available for Blast searches and can be
downloaded at the Broad Institute Web site.
The Broad assembly was compared to Bayer’s
assembly for validation and assignment of 35
supercontigs (98% of the assembly) to chromosomes. As a biotrophic pathogen, U. maydis could potentially be used as a model to
study genetically intractable but important
crop pathogens, such as the rust and bunt
fungi. Comparative analysis with the Puccinia
graminis genome, when it is released, will be
very useful to study the evolution of the smut
and rust fungi. For well-conserved genes, U.
maydis can serve as a surrogate model for the
functional characterization of P. graminis and
other rust genes.
NSF has funded sequencing for 12X coverage of P. graminis and 40,000 ESTs from six
different EST libraries (urediospores, germinating urediospores, teliospores, aeciospores,
isolated haustoria, and infected leaves). To
date, 8X coverage of WGS sequences and
25,000 ESTs have been generated at the Broad
Institute (L. Szabo, personal communication).
The strain used for genome sequencing is
CRC75-36-700-3, which carries avirulence
loci for at least 25 wheat stem rust resistance genes. Other basidiomycete pathogens
to be sequenced at JGI include Phakopsora
meibomiae and the Asian soybean rust fungus P. pachyrhizi. Unfortunately, the estimated
genome sizes of these two Phakopsora species
are surprisingly large (about 700 Mb) and
only 1X coverage of P. pachyrhizi has been sequenced (J. Boore, personal communication).
Two saprophytic basidiomycetes, P.
chrysosporium and Coprinus cinereus, have been
sequenced. These saprophytic fungi can
degrade all components of wood, including
lignin. P. chrysosporium is used in industry for
pulp bleaching and remediation of organopollutants (87). The 30-Mb genome of P.
chrysosporium strain RP78 was sequenced at
JGI. A total of 611,025 paired end sequences
(10.6X coverage) were assembled into 232
scaffolds with a combined contig length
of 35.1 Mb. Many predicted genes encode
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secreted oxidases, peroxidases, and hydrolytic
enzymes that are involved in wood decay. C.
cinereus is a multicellular basidiomycete with
a typical mushroom form (Agaricales) that
can complete its entire life cycle within 2
weeks in the laboratory. Its haploid genome
has 13 chromosomes, ranging in size from
1–5 Mb and an estimated size of 37.5 Mb.
The genome assembly of 10X sequence
coverage and automated genome annotation
are available at the Broad Web site. Genomic
analysis of C. cinereus will provide useful
information on various aspects of mushroom
development, such as gill formation, stalk
elongation, and basidiospore discharge.
Other saprophytic basidiomycetes that are
being sequenced at JGI include Laccaria bicolor, Postia placenta, and Sporobolomyces roseus.
Multiple strains of the human pathogen C.
neoformans have been sequenced and can
be used for comparative analyses with plant
pathogenic basidiomycetes.
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Zygomycetes, Chytrids,
and Oomycetes
Although not many zygomycetes or chytrids
are important plant pathogens, studies on
these lower fungi are important to understand
genome structures and the evolution of ascomycete and basidiomycete pathogens. The
Broad Institute has sequenced and released
the genome sequence of Rhizopus oryzae. The
14.5X sequence coverage of clinical isolate RA
99–880 has been assembled into 389 contigs
in 81 supercontigs. The total length of assembled contigs is 45.3 Mb, which is larger than
ascomycetous yeasts and similar to most filamentous ascomycetes. JGI has been funded
to sequence several chytrids and zygomycetes,
including Batrachochytrium dendrobatidis, Glomus intraradices, and Phycomyces blakesleeanus.
Oomycetes and true fungi share many
common features in growth, development,
and plant infection. To date, three Phytophthora species have been sequenced. Over
75,757 ESTs and 1X coverage of P. infestans were sequenced by Syngenta (109). The
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Xu et al.
NSF/USDA Microbial Genome Sequencing Program has funded sequencing of an
additional 8X coverage of the P. infestans
genome (H. Judelson, personal communication). Genome sequencing efforts at JGI have
focused on P. sojae and P. ramorum. P. sojae, the causal agent of soybean root rot, has
an estimated genome size of 95 Mb and has
been developed as a model species for the
genus. The current draft of the P. sojae genome
had over 1 million reads assembled into 1810
scaffolds totaling over 86 Mb. This release
of 9X sequence coverage includes a total of
19,276 gene models (B. Tyler, personal communication). The Sudden Oak Death disease caused by P. ramorum is now destroying Pacific coastal oak and other trees. JGI
has sequenced 7X coverage of the P. ramorum genome. The current genome assembly
consists of 2576 contigs (66.6 Mb combined
length) and contains 16,066 predicted gene
models. The P. sojae and P. ramorum genomes
show substantial synteny except in regions
encoding putative pathogenicity genes (125).
Sequence conservation between these two
Phytophthora species has provided additional
support for gene models predicted by automated annotation.
ANNOTATION AND GENOME
STRUCTURES
Gene Annotation
Identifying gene coding sequences is an
immediate goal in any genome sequencing
project. For fungal genomes sequenced at the
Broad Institute, gene structures are predicted
using the Calhoun annotation system that
is a combination of FGENESH (http://
www.softberry.com), FGENESH+, and
GENEWISE. This approach was used for
the automated annotation of the genomes
of N. crassa, M. oryzae, F. graminearum, and
A. nidulans (20, 29, 30). According to the
published genomes, there are only 10,082
and 9457 genes in saprophytic N. crassa and
A. nidulans, respectively. The plant pathogens
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M. oryzae and F. graminearum have 11,108
(20) and 11,640 predicted genes, more than
the nonpathogens. JGI has developed its own
program for automated gene prediction. A
total of 10,048 gene models were identified
with BestModels v2.1 in P. chrysosporium.
In the recent update, JGI has revised the
protein-coding genes of the P. chrysosporium
genome to 11,777. Based on fungal genomes
that have been annotated, the percentage of
genic sequences varies from 37% to 61%, and
coding sequence lengths average between 1.3
and 1.9 kb (31).
The Munich Information Center for Protein Sequences (MIPS) has independently annotated the F. graminearum genome using
FGENESH with a matrix trained on fungal sequences of diverse origins (U. maydis,
Schizosaccharomyces pombe, and others). A total
of 14,100 genes were predicted by MIPS (43),
indicating that significant discrepancy exists
between different automated gene prediction
systems. Comparison of orthologous genes in
A. fumigatus with those of other Aspergillus
species revealed numerous examples of nonidentical gene models generated by independent annotation programs (30). However, this
problem is not unique to fungal genomes, although we expect that annotation in fungi will
be more accurate with their relatively simple gene structures. Even in model organisms,
for which most of the gene-calling programs
were developed, de novo gene prediction has
been problematic (11). Both FGENESH and
FGENESH+ utilize a statistical model of
gene structure that requires training on each
organism for accurate prediction. Expressed
sequence tag (EST) data sets will be helpful to
better train these programs and improve gene
prediction. However, most fungal pathogens
lack large EST data sets. To improve gene prediction in F. graminearum, the Broad Institute
has sequenced an additional 25,000 ESTs. In
the latest release of the N. crassa (release 7)
and M. oryzae (release V2.2) genomes, 10,620
and 12,841 genes were predicted, respectively,
with modified, less conservative gene prediction guidelines at the Broad Institute. A simi-
lar revision in the number of predicted genes
is likely in the next release of the F. graminearum genome. As more fungal genomes are
sequenced and analyzed, we expect that the
accuracy of automated gene prediction will
improve. Community-based manual annotation will certainly be another approach to improve annotation, but that will be a long-term
project.
When genome sequences become available for closely related species, comparative
gene prediction will be useful to improve the
accuracy of automated annotation since de
novo gene prediction is not reliable. Using
comparative annotation of four closely related Saccharomyces species, Kellis et al. (67)
revised the gene count in S. cerevisiae and
identified a large number of new regulatory motifs. About 15% of the previously
predicted ORFs in S. cerevisiae were found
to be improperly annotated (67). When the
TWINSCAN gene prediction algorithm was
adapted and used to analyze two closely related strains of C. neoformans, approximately
60% of known genes were predicted correctly
at every coding base and intron splice site. For
previously unannotated TWINSCAN predictions, over 70% were confirmed by RTPCR and direct sequencing (119). However,
most plant pathogenic fungi that have been or
are about to be sequenced are not as closely
related. Even for the Fusarium species being
sequenced, they, similar to three published Aspergillus species, may be too distantly related
for deeper comparative genomic analyses.
Common, Unique Fungal Genes
The Kingdom Fungi contains a diverse group
of eukaryotic organisms with different habitats and life styles. Approximately 30% of
the predicted genes for filamentous fungi sequenced to date have no significant homologs
in other organisms. Therefore, it may be difficult to identify a set of fungal-specific genes.
When compared with genomes of 13 other
fungi, 3340 yeast genes had homologs in at
least 12 of them. Only 17 of these “common”
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fungal genes had no significant homologs
in other organisms (49). Five are hypothetical proteins of unknown function, and the remainder are involved in various cellular processes. Although more study is required, this
small subset of genes may be involved in
fungal-specific processes.
Most plant pathogenic fungi are filamentous ascomycetes or basidiomycetes. However, it may be also difficult to identify
genes that are common but unique to all
filamentous fungi and that function as major determinants of polarized hyphal growth.
The filamentous fungus Ashbya gossypii has a
genome of 9.2 Mb, smaller than that of S. cerevisiae (22). More than 90% of 4718 predicted
A. gossypii genes are conserved in sequences
and gene order with their orthologs in S. cerevisiae. Despite their differences in morphology and life styles, A. gossypii and S. cerevisiae
share highly conserved syntenic homologs of
all known cytokinesis, cell cycle, and cell separation genes (104). Yeast certainly contains
all the genes essential for hyphal growth in
A. gossypii. Differences in gene expression or
regulation are apparently responsible for the
lack of functional conservation of highly homologous genes and for distinguishing the
yeast form from hyphal growth. In contrast
to yeasts, A. gossypii does not have any transposons, which might contribute to differences
in regulation of many genes.
In A. nidulans, A. oryzae, and A. fumigatus, comparative analysis has identified subsets of genes unique to each species that may
be at least partially responsible for the differences between them (30, 86, 97). However, it
may be impossible to identify a set of genes
specific to and common in Aspergillus species
that span over 200 million years of evolution.
The teleomorphs of some Aspergillus species
actually belong to different genera. For example, A. fumigatus is more closely related to
A. fischerianus (teleomorph Neosartorya fischeri)
than to A. nidulans or A. oryzae. Among 9926
predicted genes in A. fumigatus, the smallest sequenced Aspergillus genome, 700 are
absent or significantly diverged in N. fischeri
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(97). Over half of these are of unknown function and some have temperature-dependent
expression patterns and may be important for
virulence.
Introns
Fungal genes have relatively simple structures, which facilitates the accurate prediction
of intron boundaries. In hemiascomycetous
yeasts, intron loss appears to be dominant
during their evolution although intron gain
and conservation also occur (8, 110). To investigate intron conservation in four Euascomycetes, A. nidulans, F. graminearum, M.
oryzae, and N. crassa, a probabilistic model
was developed to estimate the most likely
rate of intron gain and loss that gave rise
to the observed intron conservation patterns
(3450 intron positions) in 2073 putative orthologs (96). Surprisingly, all three fungi had
significant numbers of intron gains compared to A. nidulans as an outgroup. The
gained introns have consensus terminal dinucleotides (GT. . .AG) and a putative branch
point sequence that matches the yeast consensus (TACTAAC) at six of seven positions
(96). Rates of intron gain varied substantially
between gene families. The number of intron gains in the PRPP synthetase gene is 11
in M. oryzae and 6 in N. crassa, significantly
higher than the average for other genes analyzed. The numbers of gained and lost introns
are approximately balanced in M. oryzae and
F. graminearum. In N. crassa, roughly twice as
many introns are lost as gained. These analyses indicate that intron gain is as significant
as intron loss in fungi (96). Introns are usually
biased toward the 5 -ends of genes in intronpoor genomes (such as many ascomycetes) but
are evenly distributed in intron-rich genomes
(93). Current models attribute this bias to 3 intron loss through a polyadenosine-primed
reverse transcription mechanism. However,
contrary to what would be expected with these
models, the rate of intron loss tends to be
lower rather than higher at the 3 -ends of
genes, suggesting that either other mutational
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mechanisms or the presence of selective pressure to preferentially conserve introns near
the 5 - and 3 -ends of genes (96).
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Microsynteny
Conservation of genomic regions and organization has been observed in closely related
Aspergillus species and between N. crassa and
Sordaria macrospora (30, 99). Analysis of orthologous pairs of genes in M. oryzae, F.
graminearum, and N. crassa revealed that syntenic regions between these fungi usually are
small, ranging from 3 to 20 genes. Based on
the MIPS analysis, there are 359, 258, and 86
regions containing four or more genes that
are colinear between F. graminearum and N.
crassa or M. oryzae or A. nidulans, respectively
(Table 2). F. graminearum and N. crassa apparently share more microsyntenic regions than
other fungi. No microsyntenic regions that
contain more than seven genes are conserved
among N. crassa, F. graminearum, A. nidulans,
and M. oryzae except the quinate/shikimate
(Qa) metabolic pathway gene cluster. This
seven-gene cluster, spanning about 20 kb on
chromosome 3 in M. oryzae (20), is absent in
S. cerevisiae and S. pombe.
Although no clear relationship could be
established between the chromosomes of F.
graminearum, N. crassa, and M. oryzae, it appears that certain chromosomal fragments
are conserved among them. All 21 syntenic
blocks identified between chromosome 7 of
M. oryzae and the N. crassa genome were
found on N. crassa chromosome 1 (122), but
their relative order was not conserved between these two chromosomes. Similarly, 14
syntenic blocks were identified between chromosome 7 of M. oryzae and chromosome 2 of
F. graminearum.
Repetitive Sequences
The abundance of repetitive sequences varies
significantly in fungi. In M. oryzae, 9.7%
of the genome assembly comprises repetitive DNA sequences longer than 200 bp and
with greater than 65% similarity (20). Most
of these repetitive sequences belong to five
retroelements and three DNA transposons.
In contrast, repetitive sequences account for
Table 2 Syntenic regions conserved between Fusarium graminearum and
three other filamentous ascomycetes
Number of genes per
syntenic block
Number of syntenic blocks between
F. graminearum and
N. crassa
M. oryzae
A. nidulans
3∗
268
251
168
4
137
114
50
5
89
56
22
6
59
44
7
7
25
22
1
8
23
8
3
9
14
5
2
10
7
4
11
4
3
12
20
4 or more
1
1
1
358
258
1
254
∗
Based on synteny analysis data available from the MIPS (http://mips.gsf.de/genre/proj/
fusarium). The comparison is based on genome wide protein BlastP searches. The selected
maximum gap between genes belonging to one syntenic region is 4 genes.
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less than 4% of the N. crassa and A. nidulans
genomes (29, 30). F. graminearum has considerably fewer repeats. Only a very small portion
(<0.5%) of the F. graminearum assembly is
repetitive sequence. There are a few Fot1- and
Fot5-like elements (18), but they all have truncated transposase genes. Comparative analysis
of simple sequence repeats (SSRs) in sequenced fungal genomes, including F. graminearum, M. grisea, N. crassa, and U. maydis,
revealed that the occurrence, relative abundance, and relative density of SSRs decreased
as the repeat unit increased (64). Mononucleotide, dinucleotide, and trinucleotide repeats are more abundant than the longer repeated SSRs.
Repetitive elements are not uniformly distributed in the M. oryzae genome. In many
cases, transposable elements are inserted into
copies of themselves or other repetitive elements. On chromosome 7, transposable elements are largely restricted to three clusters
located in chromosomal segments that have
a high recombination rate (122). These clusters are marked by more frequent gene duplications, and genes within the clusters have
greater sequence diversity than orthologous
genes from other fungi. The M. oryzae
genome contains many copies of full-length
sequences of transposable elements, such as
Pyret and Pot2 (20). In the 1.6-Mb minichromosome of the Japanese field isolate 949009,
at least three new classes of full-length retrotransposons have been identified (117).
As one of the genome defense mechanisms
against invading sequences or amplification of
selfish DNA (32), repeat-induced point (RIP)
mutation occurs with duplicated sequences
introduced by transformation in M. oryzae
(53) and F. graminearum (H. Kistler, personal
communication). Orthologs of the RID (RIP
defective) DNA methyltransferase gene required for RIP in N. crassa are present in all
phytopathogenic ascomycetes that have been
sequenced to date, including M. oryzae and F.
graminearum, but not in U. maydis or R. oryzae.
Genome-wide analysis of repetitive elements
also indicates that RIP may be responsible
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Xu et al.
for sequence variation among the repetitive
sequences. However, RIP in M. oryzae appears to be less efficient than in N. crassa (20,
53). The tolerance to highly similar repetitive sequences also can be attributed to the
asexual reproduction lifestyle of M. oryzae
field strains. In contrast, ascospores are the
primary inoculum for the wheat scab fungus (40). Frequent sexual reproduction during
the infection cycle may allow F. graminearum
to prevent the amplification of transposable elements and remove highly repetitive
sequences.
Telomeres and Telomeric Regions
Like other eukaryotes, the ends of fungal
chromosomes consist of tandem arrays of simple sequence repeats that are usually GTrich. The most common telomeric repeat in
filamentous fungi is (TTAGGG)n (91). In
most cases, telomeres are not clonable and
therefore not included in genome assemblies.
Methods have been developed (80) to identify
telomere sequences in various fungi (http://
www.genome.kbrin.uky.edu/fungi tel). By
identifying and sequencing fosmid clones with
telomeric repeats, the sequences of all 14
chromosome ends have been generated in
M. oryzae (M. Farman, personal communication). Eleven chromosome ends have the
same basic organization, with each containing a telomere-linked RecQ helicase (TLH)
gene that is 2.2–5.5 kb away from the telomere repeats (20–30 copies of TTAGGG). The
TLH genes are ubiquitous among M. oryzae
strains isolated from rice but not from other
host plants (34). Similar telomere-linked helicase genes have been found in many other
fungi, including Metarhizium anisopliae (54), F.
graminearum, S. cerevisiae, and U. maydis but
not in F. verticillioides, N. crassa, or B. cinerea
(M. Farman, personal communication). Although the function of these TLH genes is
not clear, they are apparently not important
for fungal pathogenesis.
In the current assembly of F. graminearum, no contig contains telomeric repeats.
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However, a total of 108 excluded reads have
at least five tandem copies of TAAGGG.
One in planta expressed gene identified by
subtraction, Fgr-S4 3 M04, was found to be
located near the telomere (41). This gene
was not predicted by automated annotation
in F. graminearum. A 215-bp sequence of this
gene is repeated 29 times in the genome,
and the copies are preferentially located near
telomeres (41). This repeat does not encode
any ORF larger than 60 amino acids but its
core repeat region is homologous to the long
terminal repeat (LTR) of the retrotransposon Skippy from F. oxysporum (18). Among
113 nontransposon-related genes identified
within 100 kb of the telomeres in M. oryzae
(M. Farman, personal communication), 23
encode putative secreted proteins that may be
involved in plant infection. Although the role
of these genes in fungal pathogenesis needs
to be functionally characterized, the dynamic
nature of telomeres could generate variation
in genes critical for fungal-plant interactions.
In M. oryzae, a few avirulence genes map to
telomeric locations and are unstable (127).
The 3 -UTR of the first cloned Avr-Pita allele is only 48 bp away from the telomeric
repeats (102). The sequenced M. oryzae strain
70–15 contains two putative Avr-Pita alleles
but none of them is near telomeres.
PATHOGENICITY-RELATED
GENES
Genes Unique to M. oryzae and/or
F. graminearum
Although A. gossypii has a smaller genome than
S. cerevisiae, it is a weak pathogen on cotton. This fact may argue against the presence of a core set of genes that are specific
to plant pathogens and confer general fungal
pathogenicity. However, it remains possible
that each pathogen or pathogen group may
have its unique pathogenicity-related genes
that are adapted to specific host plants or
life styles. Approximately 30% of the predicted genes of M. oryzae and F. graminearum
have no significant homologs in other organisms and appear to be unique to these two
plant pathogenic fungi. A similar percentage
of predicted genes, however, are specific to
the saprophytes N. crassa and A. nidulans. By
BlastP searches, 145 F. graminearum genes
have homologs in M. oryzae but not in N.
crassa or A. nidulans (J. Walton, personal communication). Appropriately, 23% (34/145) of
these genes can be grouped into 14 gene families with more than two members in both M.
oryzae and F. graminearum. However, most of
these genes encode proteins of unknown function and have no homologs in GenBank.
Preliminary analysis indicated that most
known fungal pathogenicity factors in M.
oryzae or F. graminearum have homologous
genes in N. crassa and/or A. nidulans. The only
exceptions are the M. oryzae avirulence genes,
such as Avr-CO39, that have unknown biochemical functions. However, some of these
pathogenicity factor genes, such as ACE1 and
ABC1 in M. oryzae or TRI5 in F. graminearum,
belong to large families. Therefore, genome
sequencing itself is not sufficient to identify pathogenesis-related genes or genes required for pathogenicity. Nevertheless, when
genome sequences of closely related fungal pathogens become available, comparative genomic analysis in combination with
expression profiling still have great potential to address some fundamental questions in
plant pathology, including the genetic bases
for necrotrophic or biotrophic growth, host
range restriction, or tissue specificity.
Both the F. graminearum and M. oryzae
genomes contain over 30 predicted genes that
are homologous only to bacterial genes and
lack introns. Although the origin of these
genes is not clear, lateral transfer of the betaglucuronidase (gus) gene from bacteria to
fungi has been published (133).
Genes Involved in Race-specific
Interactions
To date, M. oryzae is the only sequenced plant
pathogen that has been extensively studied
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for avirulence (AVR) genes. For F. graminearum and all other sequenced necrotrophs,
there are variations in virulence among
different isolates but no race specificities. Several avirulence genes in M. oryzae have been
cloned, including PWL2, AVR-Pita, AVRCO39, and ACE1. AVR-Pita encodes a putative neutral zinc metalloprotease that may
directly interact with the product of the resistance gene Pi-ta (57). PWL1, PWL3, and
PWL4 are PWL2 homologs that have different sequence identities and chromosome locations (63). The sequenced M. oryzae strain
70–15 contains four known avirulence genes,
AVR-Pita, ACE1, PWL2, and PWL3 (20), but
not PWL1, PWL4, and AVR1-CO39. For over
40 known major rice blast resistance genes,
corresponding avirulence genes have been
mapped in M. oryzae (23, 126). However, it
is impossible to systematically search for AVR
genes in the genome sequence because of the
lack of common structural features or conserved domains among them. The M. oryzae
genome does not have orthologs of AVR genes
from other pathogenic fungi, including Avr2,
Avr4, Avr9, ECP2, ECP3, and ECP5 from Cladosporium fulvum and NIP1 from Rhynchosporium secalis (20). Similarly, F. graminearum
lacks orthologs of known M. oryzae AVR genes
such as PWL2 and AVR-CO39. The lack of sequence similarity or conservation in the fungal
AVR genes may indicate that they are not important virulence factors conserved in many
plant pathogens, or their role in plant infection is highly specialized.
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Cell Wall–and Cutin-Degrading
Enzymes
For many plant pathogens, cell wall–
degrading enzymes (CWDE) and cutinases
play important roles in penetration and colonization of plant tissues. Even in fungi like M.
oryzae that rely primarily on appressorial turgor pressure for penetration (19), hydrolytic
enzymes may facilitate plant infection by
modifying the plant surface or degrading host
cell walls around the penetration pegs (73).
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Xu et al.
Several expanded gene families in M. oryzae
encode putative cell wall- and cutin-degrading
enzymes (20). The N. crassa genome contains no cutinase gene. Some of the putative cutinase genes in M. oryzae are significantly up-regulated during infection (20).
However, determining the importance of individual CWDE and cutinase genes in plant
pathogens is complicated by their genetic redundancy and variable regulation (124, 136).
Further studies, such as the characterization
of the SNF1 homolog of C. carbonum (124),
are necessary to clarify the role of these hydrolytic enzymes during plant infection. Similarly, expansion of the CWDE and cutinase
gene families has been observed in F. graminearum, which has 6 putative cutinase and 9
putative xylanase genes (Table 3). The M.
oryzae genome has only three pectate lyase
genes and lacks recognizable pectin lyase. As a
necrotrophic pathogen, F. graminearum contains at least 13 pectate lyase and 4 pectin
lyase genes. The biotrophic pathogen U. maydis has at least three cutinase, three xylanase,
one pectin lyase, but no pectate lyase genes
(Table 3).
Secretome
Various secreted proteins likely play important roles during fungal-plant interactions. In
the M. oryzae genome, over 700 proteins are
predicted to be secreted (D. Ebbole, personal
communication), considerably more than predicted in N. crassa or A. nidulans. Furthermore, 163 of these putatively secreted proteins are in families containing at least twice
as many members as the corresponding families in N. crassa (20). An example of an expanded family in M. oryzae is one that contains
21 putative secreted proteins containing the
novel variant cysteine pattern CX7 CCX5 C.
This pattern exists only eight times in A. nidulans, four times in N. crassa, and not at all
in S. cerevisiae (20). The CBP1 gene of M.
oryzae contains two CX7 CCX5 C patterns and
is specifically expressed in germ tubes. Deletion mutants have abnormal appressorium
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Gene families related to cell wall and cutin degradation
Plant pathogenic fungi
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Gene family
Saprophytic fungi
F. graminearum
M. oryzae
U. maydis
N. crassa
A. nidulans
Cutinase
6
8
3–4
0
4
Xylanase
9
10
3
6
5
Pectin lyase
4
0
1
0
5
Pectate lyase
13
3
0
2
8
IPR001002 (chitin-binding type 1)
14
18
1
5
13
IPR000070 (pectin esterase)
3
1
2
0
2
IPR006584 (cellulose binding type IV)
1
2
0
0
0
41
42
27
31
26
IPR008985 (concanavalin A-like
lectin/glucanase)
differentiation on artificial surfaces but produce normal, functional appressoria on the
leaf surface (62). In C. fulvum, Avr4 is a
chitin-binding protein with similar cysteine
patterns (128). In the genome sequences
of other plant pathogenic fungi such as
F. graminearum, at least 15 genes contain
the chitin-binding motif (IPR001002). However, none of them has been functionally
characterized.
Another expanded family of secreted proteins is similar to the necrosis-inducing peptide of P. infestans (NPP1, IPR008701), which
may function as putative effector in fungal
pathogens (9). The M. oryzae and F. graminearum genomes each contain four predicted
proteins with this domain, which is absent
in S. cerevisiae and is present in only one N.
crassa protein. Other expanded families in F.
graminearum and M. oryzae include putative
subtilisin, secreted (sf1 and sf2) protease K
(50), and cytochrome P450 mono-oxygenase
genes. The M. oryzae and F. graminearum
genomes contain 15 and 3 putative class I subtilisin genes, respectively. All of them have a
signal peptide. A. nidulans and S. cerevisiae lack
any subtilisin genes but N. crassa has one (50).
In F. graminearum, five of the P450 genes are
specifically expressed under trichotheceneproducing conditions and are unique to toxinproducing strains (123).
No special protein delivery apparatus for
transporting pathogenicity factors into plant
cells, similar to the type III secretion system
in bacteria, has been characterized in fungi.
For hemibiotrophic pathogens like M. oryzae,
some proteins must be able to pass through
the plant cytoplasmic membrane. In Uromyces
fabae, a fungal protein specifically expressed
during infection has been shown to enter host
cells and localize to plant nuclei (69), but it
has no homolog in M. oryzae and F. graminearum. Currently, it is impossible to predict
which secreted proteins have the potential to
enter plant cells. In M. oryzae and F. graminearum, 117 and 125 putative secreted proteins,
respectively, have putative nuclear localization signals (NLS). Some of these genes are
unique to M. oryzae and/or F. graminearum.
One of them is FG11447, which was found
to be important for virulence in F. graminearum in preliminary studies (X.H. Zhao &
J.-R. Xu, unpublished). However, FG11447
has no known homologs in GenBank and its
annotation is different between the Broad Institute and MIPS and needs to be experimentally validated.
The cAMP Signaling and MAP
Kinase Pathways
In several phytopathogenic fungi, the cAMP
signaling and two MAP kinase pathways
have been implicated in regulating various plant infection processes (78, 137). In
M. oryzae, PMK1 and MPS1 are two MAP
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kinase (MAPK) genes that regulate appressorium formation and penetration, respectively, and are essential for plant infection. A
third MAPK gene, OSM1, is important for
osmoregulation in vegetative hyphae but dispensable for pathogenesis (138). Similar to
the PMK1 and MPS1 homologs in several
other fungal pathogens (137), GPMK1 and
MGV1 of F. graminearum also are required for
wheat infection (48, 56). The F. graminearum
genome contains one ortholog of OSM1,
which has not been characterized. F. graminearum and M. oryzae have three MAPK kinase (MEK) and three MEK kinase (MEKK)
genes, which likely form three MAPK cascades similar to yeast Fus3/Kss1, Hog1, and
Slt2 pathways (20, 141). Several additional
components, including Cdc42, two PAK kinases, and Ste50, have also been identified
based on their yeast homologs (20). However,
the signal inputs and outputs of these three
MAPK pathways in fungal pathogens must be
different from those in yeast. Some of these
conserved genes, such as the Ste20 homolog
in U. maydis and M. oryzae (79, 114), may regulate various biological processes in different
fungi. The M. oryzae, N. crassa, and F. graminearum genomes have no putative homolog of
yeast Ste5, a scaffold protein conferring pathway specificity. In the basidiomycete U. maydis, Kpp2 and Kpp6 are two overlapping MAP
kinases involved in mating and plant infection.
However, this situation appears to be unique
to U. maydis because Kpp6 is an unusual fungal MAP kinase (10).
Putative key components of cAMP signaling, including the trimeric G proteins, adenylate cyclase, regulatory subunit, and two catalytic subunits of protein kinase A (PKA), and
two Ras proteins, also are well conserved in M.
oryzae, F. graminearum, and other sequenced
phytopathogenic fungi. In M. oryzae, cAMP
signaling has been characterized for its role
in surface recognition and initiation of appressorium formation. Although it is likely
to be important for pathogenesis, the role of
cAMP signaling has not been determined in
F. graminearum. In Cryphonectria parasitica, the
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BDM1 (beta disruption mimic factor-1) gene
is involved in G-protein signaling (65). Orthologs of BDM1 are found in M. oryzae and
F. graminearum. The TBL1 gene encodes a
transducin-beta (G-beta)-like protein and is
essential for plant infection in F. graminearum
(113).
Signal Receptors
During different infection stages, fungal
pathogens must be able to recognize various signal molecules or ligands from plant
cells. Among three major classes of receptors known in eukaryotes, the G protein–
coupled receptors (GPCRs) are the biggest
group involved in recognizing diverse external signals and regulating different cellular
processes by association with heterotrimeric
G proteins (81). In S. cerevisiae, three known
GPCRs, Ste2, Ste3, and Gpr1, are important for perception of pheromones and carbon
source and play critical roles in mating and
filamentous growth. The M. oryzae genome
contains a large number of GPCR-like genes
(76), including putative homologs of known
fungal GPCRs, such as GprD and Pre-1,
and the cAMP receptors from Dictyostelium
discoideum (71, 129). Twelve of these putative GPCR genes form a subfamily and contain an N-terminal extracellular membranespanning domain (CFEM) that is unique to
filamentous ascomycetes. A member of this
new class, PTH11, is involved in surface
recognition during appressorium formation
and is required for pathogenesis (21).
The F. graminearum genome also contains
a large number of putative GPCR proteins.
However, it has only five putative CFEMGPCR genes. The CFEM-GPCRs are novel
and unique to filamentous ascomycetes in
the Pezizomycotina. Homologs of PTH11
have not been identified in yeast and basidiomycete genomes that have been sequenced.
These putative GPCRs may be involved in
recognizing environmental and physiological
signals or adjusting to in planta conditions.
However, predicting GPCRs is not reliable
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and it is not clear whether these genes are true
orphan GPCRs. The F. graminearum genome
has six copies of IPR003014 N/apple PAN and
four copies of IPR009030 growth factor receptor domains. These domains are absent in
M. oryzae, N. crassa, and A. nidulans. It will be
interesting to determine the functions of these
putative F. graminearum–specific receptors.
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PKS and NRPS Genes
Phytotoxic metabolites produced by fungal
pathogens play important roles in plant infection (66, 135). Polyketides constitute one
major class of phytotoxins and mycotoxins, such as AAL toxin and fumonisins. In
F. graminearum, all predicted 15 polyketide
synthases (PKS) genes have been functionally characterized (28). Five of these genes
are responsible for producing the mycotoxins zearalenone, aurofusarin, and fusarin C
and the black perithecial pigment. Although
secondary metabolism is not well studied in
M. oryzae, several phytotoxic polyketide compounds are produced in cultures and melanin
in appressoria is synthesized from a polyketide precursor (100, 126). The M. oryzae
genome contains 23 putative PKS genes (20).
Other sequenced plant pathogens, including B. cinerea, S. nodorum, S. sclerotiorum,
and F. verticillioides, also have over 15 putative PKS genes (75). Fungal PKS genes vary
significantly, and even closely related fungal genomes share only a few putative orthologous PKS genes. The diverse sequence
and domain structure of PKS genes enable
phytopathogenic fungi to synthesize a variety of polyketide metabolites that may be
involved in fungal-plant interactions. In F. verticillioides, most of the genes involved in fumonisin synthesis, including the FUM1 PKS
gene, form a cluster (108). The entire FUM
cluster is absent in F. graminearum, but its immediate upstream and downstream flanking
sequences are conserved between these two
species (131).
Some small peptides synthesized by nonribosomal peptide synthetases (NRPS) also are
important virulence factors. The M. oryzae
genome contains six predicted NRPS genes
and eight putative hybrid PKS-NRPS genes
(20). A total of 20 NRPS and PKS-NRPS
genes have been identified in F. graminearum.
In M. oryzae, the avirulence gene ACE1 encodes a hybrid PKS-NRPS and is specifically expressed in late stages of appressorium
formation (6), suggesting that the secondary
metabolite(s) synthesized by Ace1 must be
able to enter plant cells for the race-specific
interaction. In Cochliobolus heterostrophus and
F. graminearum, one NRPS gene, NPS6, is
important for plant infection (77). Another
predicted NRPS, CPS1, is also important
for virulence in C. heterostrophus and other
fungi (84). CPS1 and NPS6 orthologs exist in
M. oryzae, but their functions are not clear.
The sirodesmin biosynthesis cluster of Leptosphaeria maculans is also conserved in several
filamentous ascomycetes (35).
FUNCTIONAL GENOMIC
STUDIES
To understand molecular mechanisms of fungal pathogenesis, it is necessary to determine
the function of individual genes and genomewide networks. In the past few years, a variety of functional genomics tools and resources have been developed in S. cerevisiae
(24, 36, 37, 51), but functional genomics research in plant pathogenic fungi is still in its
infancy.
EST and Homemade Microarrays
Large-scale sequencing of ESTs is a rather
simple and inexpensive gene-discovery
method, and sequencing of nonnormalized
EST libraries is a primitive transcript profiling approach. Small to medium-size EST
databases (up to several thousand clones)
have been produced in a large number
of fungal pathogens, including M. oryzae,
F. graminearum, and F. verticillioides, and
relative abundance of ESTs has been used to
identify fungal genes differentially expressed
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during different developmental or infection
processes (115). The COGEME (Consortium for Functional Genomics of Microbial
Eukaryotes; http://www.cogeme.ex.ac.uk)
database currently hosts over 54,000 ESTs
from 13 fungal species and 2 Oomycete plant
pathogens as well as suitable bioinformatics
tools (116). Over a dozen genes highly and
specifically expressed during appressorium
formation in M. oryzae, including MPG1,
UVI-1, GAS1, and GAS2, have been identified. Several genes with enhanced expression
in germlings of the pmk1 mutant may be related to protein and melanin synthesis (116).
Before genome sequences were available,
ESTs were used to generate macroarrays
(high-density membranes of cDNA clones)
and spotted cDNA microarrays (cDNA fragments spotted on glass slides) in several plant
pathogenic fungi, including C. parasitica, M.
oryzae, and F. verticillioides (1, 106). However,
arrays of this type have marginal reproducibility and sensitivity, and they represent
only a fraction of the fungal genome.
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Whole-Genome Microarrays
Whole-genome microarray experiments allow genome-wide monitoring of transcript
abundance. In S. cerevisiae, expression profiling has identified transcription regulatory
motifs and networks (52, 85). The tiled microarray approach also has been used to
study transcription regulatory elements, chromatin structure, and nucleosome positioning (38, 140). In C. albicans and C. neoformans, microarray analysis has been applied
to study drug resistance, cell wall synthesis,
temperature shifts, and pathogenic development (26, 82). For plant pathogenic fungi,
whole-genome microarrays are now available
for M. oryzae (http://www.agilent.com) and
F. graminearum (Affymetrix). Microarrays of
U. maydis, A. flavus, and F. verticillioides are
being developed.
The M. oryzae array contains 13,666 fungal elements representing the Broad Institute predicted gene set, additional predicted
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features from other gene models, and ESTs
of unpredicted genes. This array has been
used to identify genes differentially expressed
during spore germination and appressorium
formation (20), in different plant infection
stages, and in cultures under various nutritional stresses. About 2% and 4% of genes
were differentially regulated in immature
(7 h) and mature (12 h) appressoria, respectively, compared with spores germinated on a
noninducive surface. Additional information
about these gene-profiling experiments can be
found at MGOS (http://www.mgosdb.org).
Under appressorium-inducing conditions,
the pmk1 and mst12 mutants defective in appressorium formation and penetration (13,
103) exhibited differential expression of about
300 genes compared to the wild type. Other
transcriptional profiling experiments have focused on in planta gene expression. At 48-h
post-inoculation (hpi), the expression of 17
fungal genes could be detected in infected barley leaves, which rose to 348 genes at 96 hpi.
Analysis across all microarray experiments to
date has revealed that about 60 genes are
specifically expressed in planta, including several transporters and proteins involved in xylan and lipid metabolism (R.A. Dean, personal
communication).
The F. graminearum GeneChip microarray
has 18,069 probe sets, including a combined
set of 16,926 genes calls (Broad + MIPS) and
611 ESTs (44). Each probe set is generally
represented by 11 pairs of 25-bp primers. Hybridization experiments using genomic DNA
has demonstrated the usefulness of the array
for F. graminearum and at least four closely related Fusarium species (F. asiaticum, F. boothii,
F. culmorum, and F. pseudograminearum). Differential transcript accumulation was detected
in F. graminearum grown under three nutritional conditions and in infected barley samples (44). The ability to detect fungal genes
in planta is surprisingly sensitive even without
enriching for fungal transcripts. Raw and normalized expression data with barley samples
infected with F. graminearum collected at 24,
48, 72, 96, and 144 hpi have been deposited
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at the Plant Expression Database (PLEXdb,
http://www.plexdb.org). Similar infection
time-course experiments are in progress with
flowering wheat heads inoculated with two F.
graminearum strains of different virulence (H.
Kistler, personal communication). Other ongoing gene expression profiling experiments
include different developmental and infection
mutants, such as the gpmk1, mgv1, and tbl1
mutants.
SAGE and MPSS
Serial Analysis of Gene Expression (SAGE)
has been applied to fungal pathogens (55,
121). In M. oryzae, 57 and 53 genes were found
to be up- and downregulated by cAMP treatment, respectively, by SAGE analysis with
mRNA isolated from conidia germinating
in the presence or absence of cAMP (55).
Many of these cAMP-induced genes have no
homologs in GenBank, but some of them
are well-characterized pathogenicity factors,
such as MPG1, GAS2, and MAC1. Modified
SAGE methods, such as Robust LongSAGE
(RL-SAGE) and SuperSAGE, also have been
used to study M. oryzae-rice interactions (42,
88). Among 12,119 SuperSAGE tags obtained
from M. oryzae-infected rice leaves, 74 (0.6%)
are derived from fungal genes. The most
abundantly expressed gene is MPG1, which
accounted for 38 tags. PUB4 and a nucleosidediphosphate kinase gene are among the other
M. oryzae genes highly represented by SAGE
tags (88). Massively Parallel Signature Sequencing (MPSS) is another gene expression
profiling method, but to date there is no
published MPSS analysis in phytopathogenic
fungi.
Targeted Mutagenesis
In all fungal pathogens sequenced to date,
approximately 30% of predicted genes do
not have known homologs (31). Determining the function of individual genes by systematic targeted mutagenesis is important.
In S. cerevisiae, a collection of deletion mu-
tants with unique sequence tags has been
generated for nearly every (96%) predicted
gene (37). Unfortunately, due to the relatively low efficiency of homologous recombination in most filamentous fungi, flanking
sequences longer than 0.5 kb are required to
obtain 5%–10% gene replacement transformants. Therefore, the direct PCR approach
developed for yeast knockout analysis (5) is
not applicable to fungal pathogens. Several
strategies have been developed to improve the
generation and screening of gene knockout
strains. The transposon-arrayed gene knockout (TAG-KO) technology has been used to
generate a large collection of disruption vectors by in vitro insertional mutagenesis in
M. oryzae and M. graminicola (45), but the
collection is not available to the public. Recently, several approaches have been developed to improve the efficiency of constructing gene replacement cassettes (132, 139, 142)
and screening for knockout mutants in filamentous fungi (15, 70). The split-marker approach (15) significantly increases the percentage of gene replacement transformants
in several fungi tested, including F. graminearum and C. heterostrophus, but it has no obvious effect in M. oryzae (J.-R. Xu, unpublished). Filamentous fungi contain homologs
of human Ku70 and Ku80, which encode proteins that function in nonhomologous endjoining of double-stranded DNA breaks. In N.
crassa, the mus-51 and mus-52 mutants deleted
of Ku70 and Ku80 homologs, respectively,
were increased significantly in the homologous recombination frequency (98). There is
an ongoing project to generate a large collection of deletion mutants with the mus-51 and
mus-52 mutants (http://www.dartmouth.
edu/∼neurosporagenome). Deletion of the
Ku70 or Ku80 homologs will probably improve the efficiency of targeted gene deletion
in plant pathogenic fungi such as M. oryzae.
However, the removal of the Ku70 or Ku80
deletion background is achieved by genetic
crosses, and it will be technically challenging
for fungal pathogens with low female fertility
or no known sexual stage.
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RNA-mediated gene silencing is also suitable for characterizing gene functions (14, 95).
Since it is more efficient than antisense RNA
at posttranscriptional gene silencing, RNA interference (RNAi) has been widely used in
plants and animals (14, 95). During RNAi,
double-stranded RNA is cleaved into small
(21–26 bp) interference RNA (siRNA), which
can be incorporated into a multicomponent
RNase complex (RISC) and direct the degradation of mRNA with homologous sequence.
In contrast to gene knockout approaches,
RNAi can silence genes to various degrees and
be used to study the function of essential genes
in haploid fungal genomes. RNAi is also more
advantageous to study genes with overlapping
functions or genes belonging to multimember families. RNAi has been used to silence
a few genes by expressing hairpin RNA constructs in several filamentous fungi, including M. oryzae, A. flavus, and F. graminearum
(59, 90, 95), but whether it is suitable for
large-scale mutagenesis in fungal pathogens
remains to be tested. Since a short fragment
of 25 bp is sufficient for silencing, specific
siRNA molecules can be synthesized, but it
may be difficult to efficiently introduce siRNA
molecules into filamentous fungi and assay for
silencing effects.
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Large-Scale Random Insertional
Mutagenesis
Random insertion transformants have been
generated in various fungal pathogens using restriction enzyme-mediated integration
(REMI), Agrobacterium tumefaciens-mediated
transformation (ATMT), or transposon tagging. To date, the most extensive collection of
mutants in plant pathogenic fungi was generated by the M. oryzae community (4, 118, 130).
Direct screens of these transformants have led
to the discovery of novel pathogenicity factors in various fungi, such as PTH11, ABC1,
and PLS1 in M. oryzae and the PIG genes in
U. maydis (60, 138). Recently, an additional
55,000 insertion mutants have been created in
M. oryzae, the majority (>40,000) by ATMT
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Xu et al.
and the remainder by protoplast transformation (M.J. Orbach, personal communication).
These transformants have been analyzed
for growth, pigmentation, and sporulation
defects and deposited at the Fungal Genetic Stock Center (http://www.fgsc.net). To
track these 55,000 transformants, a minimal
Laboratory Information Management System
(LIMS), called PACLIMS (Phenotype Assay
Component LIMS), has been developed (25).
Data can be entered into this system from separate locations to support multi-institutional
projects. In addition, the MGOS (Magnaporthe grisea-Oryza sativa) database (http://
www.mgosdb.org) has been developed as a
central repository for the rice and M. oryzae
genomic and EST sequences and data from
the mutagenesis and microarray experiments.
Similar large-scale mutagenesis projects
are being carried out by other members of the
International Rice Blast Genomics Consortium. About 100,000 ATMT transformants
have been generated with M. oryzae field isolates Y34 and P131 (Y. Peng, unpublished).
In total, over 200,000 random insertional
transformants have been generated by the M.
oryzae community. If these mutants are combined and pools of genomic DNA of these
transformants become available for mutant
screening, this will be a valuable functional
genomics resource. However, one practical
problem with distributing plant pathogens is
that permits are necessary. For other phytopathogenic fungi, such as F. graminearum,
F. oxysporum, and U. maydis, usually less than
10,000 insertional mutants are generated in
individual labs (39, 94, 113). Significant efforts are still needed to produce more random transformants. The software, database,
and experience gained from the M. oryzae
community in generating large mutant collections will be useful for similar projects in
other pathogens.
With the availability of genome sequences
and whole-genome microarrays, the signature-tagged mutagenesis (STM) approach developed for identifying survival-related genes
in bacteria also could be used to study fungal
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pathogens (83). Each STM mutant has a
unique sequence tag that can be tracked by
PCR. This approach has been used to identify virulence factors in A. fumigatus (12). In
plant pathogenic fungi, STM might be suitable to identify genes required for specific developmental or infection processes. Another
powerful high-throughput random mutagenesis approach is Targeted Induced Local Lesions in Genomes (TILLING), which consists of chemical mutagenesis followed by a
PCR screening strategy for point mutations
(89). It has been successfully used in rats,
Drosophila, and Arabidopsis (46). To our knowledge, TILLING has not been applied to any
fungal pathogen, but there is an on-going
TILLING project with P. sojae (K. Lamour,
personal communication).
PERSPECTIVES
Complete genome sequences for several
model fungal species and an increasing number of phytopathogenic fungi are now available. With decreasing cost of sequencing,
many other fungal pathogens will be sequenced in the near future and become accessible for genomic studies. As we move
further into the genomics era, data analysis rather than data acquisition will become
the rate-limiting step. To keep pace with
rapidly increasing genome sequence information, it will be necessary to develop better
bioinformatics tools. The tremendous diversity and genome flexibility in fungi, however,
will make this task difficult. For example, a
key step in sequence analysis is annotation.
Existing programs for automated gene prediction are not perfect and need to be improved
or trained better. Follow-up manual annotation also is necessary to improve the accuracy of automated annotation, but this is timeconsuming and labor-intensive. Ultimately, a
comprehensive genome database similar to
YPD (http://www.yeastgenome.org) will be
desirable for fungal pathogens.
Comparative genomics is a powerful approach to address evolutionary and phylo-
genetic questions (24, 67). In closely related
plant pathogenic fungi, comparative analysis
can be used to improve de novo gene prediction and identify genes involved in host range
determination, infection-related morphogenesis, and virulence. Sequencing of representative fungi from different phylogenetic clades
or lineages will allow kingdom-wide comparison and comprehensive analysis of fungal
genomes and their evolution (31). A Web site
(http://www.microbesonline.org) has been
developed by the Virtual Institute for Microbial Stress and Survival to include all the
prokaryotic sequences and a set of comparative genomics tools (e.g., the VertiGO Gene
Ontology browser) designed to facilitate multispecies comparison (2). Similar sites for
fungal pathogens are needed.
As an important part of functional
genomics, systematic characterization of
individual genes is necessary. However, until
more efficient gene knockout approaches become available, it is more realistic to focus on
functional characterization of candidate genes
selected by bioinformatics or expression profiling analyses. In N. crassa, a project to delete
over 100 putative zinc finger transcription
factor genes has been accomplished recently
(http://www.dartmouth.edu/∼neurospora
genome). The PKS genes in F. graminearum
and the NRPS and two-component histidine kinase genes in C. heterostrophus have
been systematically deleted (16, 28, 77).
Whole-genome microarrays of F. graminearum and M. oryzae are now available, and
can be expected for many other sequenced
fungal pathogens. For expression profiling
experiments, most bioinformatics tools and
resources that have been applied in functional
genomics studies in S. cerevisiae can be applied
to plant pathogens. However, more advanced
functional genomics tools, such as ChIP-chip
experiments (3, 47), are needed to identify
transcription factors and regulatory elements.
Application of these advanced genomics techniques will be as important as microarrays
for genomic analyses of plant pathogenic
fungi.
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Proteomics analyses are complementary to
genomics approaches (134). To date, most of
the proteomics studies in plant pathogenic
fungi have been limited to 2-D gel analysis
(72). However, various powerful proteomics
methods have been developed for genomewide analyses of protein expression, protein
localization, and protein-protein interactions
in fungi (36, 74, 105). Whole-genome protein
arrays, systematic yeast two-hybrid assays, and
a high-throughput TAP-tagging approach
have been used to characterize the yeast proteome and interactome (3, 74). Integration of
large-scale genomics and proteomics data enables the elucidation of global networks and
systems biology studies in yeast. We expect
that similar advanced proteomic resources
will soon be available for some fungal plant
pathogens.
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Overall, with more fungal genomes being sequenced, comparative and functional
genomics analyses will provide valuable
information to improve our understanding
of fungal pathogenicity genes and regulatory
mechanisms. One challenging task for the
fungal research community will be to apply
the rich information gained in genomic studies to improve crop production and agricultural practices. Another practical concern is
that advanced genomic studies may have to be
focused on a few model plant pathogenic fungi
because genomics and proteomics studies are
not cost-effective. Some important but less investigated pathogens will be further neglected
or underinvestigated. The fungal community
may have to combine resources by organizing
an international consortium with collaborations between industry and academia.
ACKNOWLEDGMENTS
We thank Drs. Larry Dunkle and Steve Goodwin at Purdue University for critical reading
of this manuscript. We also thank Drs. Jeffrey Boore, Ralph Dean, Daniel Ebbole, Mark
Farman, Scott Gold, Steve Goodwin, Barbara Howlett, Howard Judelson, H. Corby Kistler,
Kurt Lamour, Chris Lawrence, Marc-Henri Lebrun, Marc Orbach, Les Szabo, Brett Tyler,
Hans VanEtten, Jonathan Walton, and Jiqiang Yao for sharing unpublished data. Special thanks
to the editors for careful editing and thoughtful suggestions. This work was supported by a
grant from the USDA National Research Initiative to J.-R. X. and BARD grant US-3491-03
to J.-R. X. and A. S.
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Contents
Annual Review
of Phytopathology
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Volume 44, 2006
A Retrospective of an Unconventionally Trained Plant Pathologist:
Plant Diseases to Molecular Plant Pathology
Seiji Ouchi p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
The Current and Future Dynamics of Disease in Plant Communities
Jeremy J. Burdon, Peter H. Thrall, and Lars Ericson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p19
A Catalogue of the Effector Secretome of Plant Pathogenic Oomycetes
Sophien Kamoun p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p41
Genome Packaging by Spherical Plant RNA Viruses
A.L.N. Rao p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p61
Quantification and Modeling of Crop Losses: A Review of Purposes
Serge Savary, Paul S. Teng, Laetitia Willocquet, and Forrest W. Nutter, Jr. p p p p p p p p p p p p p89
Nonsystemic Bunt Fungi—Tilletia indica and T. horrida: A Review of
History, Systematics, and Biology
Lori M. Carris, Lisa A. Castlebury, and Blair J. Goates p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 113
Significance of Inducible Defense-related Proteins in Infected Plants
L.C. van Loon, M. Rep, and C.M.J. Pieterse p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 135
Coexistence of Related Pathogen Species on Arable Crops in Space
and Time
Bruce D. L. Fitt, Yong-Hu Huang, Frank van den Bosch, and Jonathan S. West p p p p p p 163
Virus-Vector Interactions Mediating Nonpersistent and Semipersistent
Transmission of Plant Viruses
James C.K. Ng and Bryce W. Falk p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 183
Breeding for Disease Resistance in the Major Cool-Season Turfgrasses
Stacy A. Bonos, Bruce B. Clarke, and William A. Meyer p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 213
Molecular Ecology and Emergence of Tropical Plant Viruses
D. Fargette, G. Konaté, C. Fauquet, E. Muller, M. Peterschmitt, and J.M. Thresh p p p 235
Biology of Flower-Infecting Fungi
Henry K. Ngugi and Harald Scherm p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 261
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ARI
1 July 2006
4:28
A Model Plant Pathogen from the Kingdom Animalia: Heterodera
glycines, the Soybean Cyst Nematode
T.L. Niblack, K.N. Lambert, and G.L. Tylka p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 283
Comparative Genomics Reveals What Makes an Enterobacterial Plant
Pathogen
Ian K. Toth, Leighton Pritchard, and Paul R.J. Birch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 305
The Dawn of Fungal Pathogen Genomics
Jin-Rong Xu, You-Liang Peng, Martin B. Dickman, and Amir Sharon p p p p p p p p p p p p p p p p 337
Annu. Rev. Phytopathol. 2006.44:337-366. Downloaded from arjournals.annualreviews.org
by TEL-AVIV UNIVERSITY on 08/08/06. For personal use only.
Fitness of Human Enteric Pathogens on Plants and Implications for
Food Safety
Maria T. Brandl p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 367
The Role of Ethylene in Host-Pathogen Interactions
Willem F. Broekaert, Stijn L. Delauré, Miguel F.C. De Bolle,
and Bruno P.A. Cammue p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 393
Phenazine Compounds in Fluorescent Pseudomonas Spp. Biosynthesis
and Regulation
Dmitri V. Mavrodi, Wulf Blankenfeldt, and Linda S. Thomashow p p p p p p p p p p p p p p p p p p p p p p 417
Long-Distance RNA-RNA Interactions in Plant Virus Gene
Expression and Replication
W. Allen Miller and K. Andrew White p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 447
Evolution of Plant Pathogenicity in Streptomyces
Rosemary Loria, Johan Kers, and Madhumita Joshi p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 469
Climate Change Effects on Plant Disease: Genomes to Ecosystems
K.A. Garrett, S.P. Dendy, E.E. Frank, M.N. Rouse, and S.E. Travers p p p p p p p p p p p p p p p p p 489
INDEX
Subject Index p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 599
ERRATA
An online log of corrections to Annual Review of Phytopathology chapters (if any, 1977 to
the present) may be found at http://phyto.annualreviews.org/
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