A phenome-based functional analysis of transcription factors in the cereal head blight fungus, Fusarium graminearum.

Abstract

Fusarium graminearum is an important plant pathogen that causes head blight of major cereal crops. The fungus produces mycotoxins that are harmful to animal and human. In this study, a systematic analysis of 17 phenotypes of the mutants in 657 Fusarium graminearum genes encoding putative transcription factors (TFs) resulted in a database of over 11,000 phenotypes (phenome). This database provides comprehensive insights into how this cereal pathogen of global significance regulates traits important for growth, development, stress response, pathogenesis, and toxin production and how transcriptional regulations of these traits are interconnected. In-depth analysis of TFs involved in sexual development revealed that mutations causing defects in perithecia development frequently affect multiple other phenotypes, and the TFs associated with sexual development tend to be highly conserved in the fungal kingdom. Besides providing many new insights into understanding the function of F. graminearum TFs, this mutant library and phenome will be a valuable resource for characterizing the gene expression network in this fungus and serve as a reference for studying how different fungi have evolved to control various cellular processes at the transcriptional level.

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A Phenome-Based Functional Analysis of Transcription
Factors in the Cereal Head Blight Fungus, Fusarium
graminearum
Hokyoung Son1., Young-Su Seo1.¤, Kyunghun Min1, Ae Ran Park1, Jungkwan Lee2, Jian-Ming Jin1, Yang
Lin1, Peijian Cao3, Sae-Yeon Hong1, Eun-Kyung Kim1, Seung-Ho Lee1, Aram Cho1, Seunghoon Lee1,
Myung-Gu Kim1, Yongsoo Kim1, Jung-Eun Kim1, Jin-Cheol Kim4, Gyung Ja Choi4, Sung-Hwan Yun5, Jae
Yun Lim1, Minkyun Kim1, Yong-Hwan Lee1, Yang-Do Choi1, Yin-Won Lee1*
1Department of Agricultural Biotechnology and Centers for Fungal Pathogenesis and Agricultural Biomaterials, Seoul National University, Seoul, Korea, 2Department of
Applied Biology, Dong-A University, Busan, Korea, 3China Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute, Zhengzhou, Henan, China, 4Chemical
Biotechnology Center, Korea Research Institute of Chemical Technology, Daejon, Korea, 5Department of Medical Biotechnology, Soonchunhyang University, Asan, Korea
Abstract
Fusarium graminearum is an important plant pathogen that causes head blight of major cereal crops. The fungus produces
mycotoxins that are harmful to animal and human. In this study, a systematic analysis of 17 phenotypes of the mutants in
657 Fusarium graminearum genes encoding putative transcription factors (TFs) resulted in a database of over 11,000
phenotypes (phenome). This database provides comprehensive insights into how this cereal pathogen of global
significance regulates traits important for growth, development, stress response, pathogenesis, and toxin production and
how transcriptional regulations of these traits are interconnected. In-depth analysis of TFs involved in sexual development
revealed that mutations causing defects in perithecia development frequently affect multiple other phenotypes, and the TFs
associated with sexual development tend to be highly conserved in the fungal kingdom. Besides providing many new
insights into understanding the function of F. graminearum TFs, this mutant library and phenome will be a valuable resource
for characterizing the gene expression network in this fungus and serve as a reference for studying how different fungi have
evolved to control various cellular processes at the transcriptional level.
Citation: Son H, Seo Y-S, Min K, Park AR, Lee J, et al. (2011) A Phenome-Based Functional Analysis of Transcription Factors in the Cereal Head Blight Fungus,
Fusarium graminearum. PLoS Pathog 7(10): e1002310. doi:10.1371/journal.ppat.1002310
Editor: Jin-Rong Xu, Purdue University, United States of America
Received June 18, 2011; Accepted August 25, 2011; Published October 20, 2011
Copyright: ? 2011 Son et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a National Research Foundation of Korea (NRF) grant (2011-0000963) funded by the Korean government (MEST) and by a
grant (CG 1141) from the Crop Functional Genomics Center of the 21st Century Frontier Research Program funded by the Korean Ministry of Education, Science,
and Technology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: lee2443@snu.ac.kr
¤ Current address: Department of Microbiology, Pusan National University, Busan, Korea
. These authors contributed equally to this work.
Introduction
Transcription factors (TFs) orchestrate gene expression under
the control of cellular signaling pathways and are key mediators of
cellular function [1]. Understanding the regulatory mechanisms of
each TF family and their function could provide valuable insight
into gene expression changes underpinning cellular and develop-
mental responses to environmental cues. Modification of TF
activity through either gene disruption or overexpression can help
determine the function and interconnectedness of individual TFs
based on resulting cellular changes.
The filamentous fungus Fusarium graminearum (teleomorph:
Gibberella zeae) is an important plant pathogen that causes head
blight of major cereal crops, such as wheat, barley, and rice [2].
The fungus also produces mycotoxins in the infected cereals,
posing grave threats to the health of animals and humans [3].
Complete genome sequencing of F. graminearum [4] has allowed for
genome-wide gene functional studies, and transcriptome data from
F. graminearum generated using the Affymetrix platform (http://
www.plexdb.org/) can be used to predict gene function based on
gene expression profiles [5,6,7,8,9,10,11,12]. In addition, TFs
from 64 fungal species, including F. graminearum, have been
annotated in the Fungal Transcription Factor Database (FTFD,
http://ftfd.snu.ac.kr/) [13]. The majority of F. graminearum TFs
belong to the Zn(II)2Cys6fungal binuclear cluster family; other
types of TFs include C2H2 zinc finger, GATA, bHLH, B-ZIP,
CBF, CCAAT-binding factor, homeobox, RING finger, PHD
finger, and MIZ zinc finger [13]_ENREF_6. Although a few TFs
that regulate pigmentation [14]_ENREF_6, mycotoxins biosyn-
thesis [15,16,17]_ENREF_7_ENREF_7, sexual development, and
virulence [18,19,20,21,22] have been characterized in F. grami-
nearum, the function and regulation of most TFs remain to be
determined.
In this study, we constructed F. graminearum deletion mutants in
657 genes encoding various types of TFs through homologous
recombination to dissect their functions; resulting mutants were
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analyzed under 17 phenotypic categories to build a comprehensive
phenotypic dataset (phenome). This phenome, which can be
accessed at FgTFPD (Fusarium graminearum Transcription Factor
Phenotype Database, http://ftfd.snu.ac.kr/FgTFPD), helped un-
derstand the mechanisms underpinning sexual and asexual
development, toxin production, stress responses, and pathogenic-
ity. The observed phenotypes were analyzed using a combination
of data from published genome-wide fungal resources and
bioinformatics analysis. Our mutant library will be a valuable
source through distribution of mutants and easy access to our
phenotypic and genetic data.
Results
Construction of genome-wide putative TF deletion
mutants in F. graminearum
In order to construct F. graminearum TF mutants, we employed
the FTFD [13] to choose the genes annotated as TFs in F.
graminearum. The FTFD is a standardized pipeline for annotating
fungal TFs using the InterPro database that is based on DNA-
binding motifs. Among the 64 classified TF families present in the
61 fungal and three oomycete species, 693 putative F. graminearum
TFs, belonging to 45 families, were annotated and selected for
deletion. We also added 16 manually identified TFs based on
sequence homology or conserved motifs (Table S1). We finally
selected 709 putative TFs, consisting of 6.1% of all the F.
graminearum genes [4]_ENREF_6. It has been reported that TFs
cover 3–6% of the genes in Drosophila melanogaster, Caenorhabditis
elegans, and Arabidopsis thaliana [23]. In the fungal kingdom the
percentage of TFs is also variable, ranging from 2.3% in
Pneumocystis carinii to 7.2% in Rhizopus oryzae [13]. Approximately
60% of the total TFs in F. graminearum belong to TFs bearing zinc
finger DNA-binding domains, including Zn(II)2Cys6, C2H2, and
CCCH (Figure 1A and Table S2). In contrast, approximately 20%
of TFs in A. thaliana and C. elegans belong to this group_-
ENREF_10. The higher percentage of zinc finger TFs in fungi is
mainly due to the existence of fungal-specific Zn(II)2Cys6zinc
finger TFs (e.g., 25% of total TFs in Saccharomyces cerevisiae) [23]. In
addition to the zinc finger-related TFs, the other major TF families
in the F. graminearum genome include OB-fold nucleic acid binding,
high mobility group, winged helix repressor DNA-binding, and B-
ZIP domains containing proteins (Figure 1A).
Using homologous recombination, we successfully disrupted
657 TF genes out of 709 (Figure 1B). Eight genes were excluded
because of failure to amplify their 59 or 39 flanking region under
various PCR conditions and primer sets. Repeated attempts to
delete the remaining 44 genes were unsuccessful likely due to
lethality. Disruption of 132 genes was confirmed by co-dominant
PCR and Southern hybridization with a 59 or 39 flanking region
probe, as previously reported [24]. The remaining mutants were
confirmed by co-dominant PCR screening followed by Southern
hybridization with a geneticin resistant gene cassette (gen) probe
(Figure S1). The FgFSR1-deletion mutant was generated in our
previous study [21]. Only co-dominant PCR screening was used to
confirm disruption of two TF mutants (FGSG_06071 and
FGSG_10716). We found an extra copy of the mutant allele
integrated ectopically in 49 mutants (Figure S1 and Table S2). We
screened more than 10,000 transformants using co-dominant PCR
to identify desired mutants, resulting in 2,183 positive mutants.
Among them, approximately 65% were single deletion mutants
without ectopic integration and 23% were deletion mutants with
multiple integrations. In a high-throughput gene deletion study in
Neurospora crassa, more than 90% deletion efficiency was obtained
using a strain with deletion of genes required for non-homologous
end-joining DNA repair (mus-51 or mus-52) [25]. The split marker
method based on triple homologous recombination to disrupt
genes in F. graminearum was efficient enough that similar mutations
of the parental strain were not required for mutagenesis.
Phenotypic analyses of resulting mutants
We analyzed 657 TF mutants for changes in 17 phenotypes
(Table S2), which fall into six major phenotypic categories
Figure 1. Classification and deletion strategy of putative
transcription factors (TFs) in Fusarium graminearum. (A) Total
TFs were classified based on nucleic acid binding domains. More than
half of the total TFs are Zn6Cys6 zinc finger and C2H2 zinc finger
proteins. (B) Each target TF gene was deleted using the split marker
method based on triple homologous recombination.
doi:10.1371/journal.ppat.1002310.g001
Author Summary
Large collections of mutant lines allow for identification of
gene functions. Here we constructed a mutant library of
657 putative transcription factors (TFs) through homolo-
gous recombination in the head blight fungus, Fusarium
graminearum, providing a resource for understanding
gene regulation in fungus. By screening these mutants in
17 phenotypic categories, we constructed a dataset of
over 11,000 phenotypes. This study provides new insight
into understanding multiple phenotypes caused by single
TF as well as regulation of gene expression at the
transcription level in F. graminearum. Furthermore, our
TF mutant library will be a valuable resource for fungal
studies through the distribution of mutants and easy
access to our phenotypic and genetic data.
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including mycelial growth (Figure S2 and Figure S3), sexual
development (Figure S4), conidia production (Table S3), virulence
(Figure S5), toxin production (Figure S6), and stress responses
(Figure S7). A substantial fraction of the mutants (26%, 170/657)
displayed clearly visible mutant phenotypes, with 73% (124/170)
of these mutants exhibiting multiple mutant phenotypes and 27%
(46/170) with single mutant phenotype (Figure 2A, Table S4, and
Table S5). Analysis of TF locations in the F. graminearum genome
revealed that the 170 TFs exhibiting phenotype changes were
randomly distributed throughout the genome (Figure 2B). Among
103 TF deletion mutants in N. crassa [25], 43% (44/103) exhibited
phenotype changes with 50% (22/44) of these exhibiting multiple
defects in growth and asexual/sexual development. Deletion of
members of the Zn(II)2Cys6TF family in N. crassa, resulted in
mutant phenotypes in 42% (30/72) of the mutants [25]. However,
we observed mutant phenotypes in only 16% (46/296) of the F.
graminearum mutants defective in Zn(II)2Cys6TFs. Deletion of the
genes encoding winged helix repressor DNA-binding TFs resulted
in the highest percentage of mutants with phenotype change (48%,
12/25), whereas Nucleic acid-binding, OB-fold TF deletion
mutants displayed the lowest percentage of phenotype change
(13%, 5/40) (Figure 2C and Table S4). The percentages of
phenotype change among the mutants of the C2H2 zinc finger,
Myb, and B-ZIP TF family members were 41% (37/91), 41% (7/
17), and 41% (9/22), respectively.
To determine correlations between phenotypes, we calculated
Pearson’s correlation coefficient (PCC) from mutants that exhibit
multiple mutant phenotypes (Table S6). The PCC between two
phenotypes was calculated to determine which phenotypes are
most highly correlated (Figure 2D). PCC.0.6 (p=3.25610212)
was chosen as the cut-off, and 18 phenotype pairs were selected
and marked with orange edges between nodes (Figure 2D). This
analysis revealed that sexual development, virulence, growth, and
toxin production were highly correlated. The representative TFs
are listed in Table 1. The phenotypes under sexual development,
including perithecia formation and maturation, and ascospore
production, were highly correlated with the phenotypes in other
categories.
We also performed in-depth studies of F. graminearum phenotypes
under diverse environmental stresses. This analysis, including
osmotic (or ionic), reactive oxygen species (ROS), fungicide, cell
wall, and acidic (pH=4) and basic (pH=11) conditions, indicates
Figure 2. Analysis of transcription factor (TF) mutant phenotypes. (A) Number of TFs showing multiple mutant phenotypes. TFs without
mutant phenotype were omitted. (B) Chromosome distribution of 709 putative TFs. TFs that exhibit mutant phenotypes (orange bars) and those that
do not (blue bars) are randomly distributed throughout the genome. Black bars represent the TF genes that could not be disrupted. (C) Number of
mutants showing mutant phenotypes in each TF subfamily. Orange and blue bars represent the number of TFs whose deletion results in a mutant
phenotype or no phenotype change, respectively. Data representing the Zn2Cys6subfamily are not shown in this graph. (D) Phenotype network
based on PCC (Table S6). PCC values were calculated from the results of tested phenotypes. Orange edges indicate positive correlations (PCC.0.60).
doi:10.1371/journal.ppat.1002310.g002
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there is no correlation between the stresses, except between
osmotic and ROS stress (Figure 2D and Table S6).
We identified the F. graminearum orthologs of the 103 TF genes
studied in N. crassa [25] to compare the resulting phenotypes
between these species (Table S7A). Among 88 F. graminearum TFs
identified, 26% of the mutants (23/88) exhibited mutant
phenotypes with 43% (10/23) of these mutants showing multiple
defects in growth and asexual/sexual development. However, only
nine mutants showed the similar phenotype changes in growth,
asexual, and/or sexual development in both species (Table S7B).
This comparison of mutant phenotypes between F. graminearum
and N. crassa suggests that the large majority of these TFs evolved
to have unique, rather than conserved, functions in controlling
growth and/or asexual and sexual developments.
Classification of TFs associated with sexual development
Among the 170 TF mutants exhibiting at least one mutant
phenotype, 105 mutants showed considerable defect in sexual
development. Based on the nature and severity of the phenotype
change in perithecia development (number of perithecia and
perithecia maturation) and ascospore production (existence and
morphology) among these 105 mutants, we classified them into
seven groups representing (Table 2 and Table S8). Four mutants
produced more perithecia than the wild-type strain (Group 1).
Group 2 contained 44 mutants that completely lack perithecia
development and could not produce any initial structure for
perithecium. The mutants showing decreased number of perithe-
cia or delayed perithecia maturation, which eventually developed
normally, were further divided into three groups based on
ascospore formation: normal-shaped ascospore formation (23
TFs, Group 3), abnormal-shaped ascospore (9 TFs, Group 4),
and no ascospore formation (19 TFs, Group 5). Among the Group
5 mutants, 12 TF mutants (GzCCAAT002, GzHMG010, MYT1,
GzOB038, FgFlbA, GzWing015, GzRFX1, GzWing027, GzZC246,
MAT1-1-3, MAT1-1-1, and MAT1-2-1) formed protoperithecia
but failed to differentiate further and the other 7 TF mutants
Table 1. List of key TF mutants showing no perithecia development and multiple defects in virulence, growth, and toxin
production.
Locus IDGene namePerithecia DevelopmentVirulenceGrowth on MMDON production
FGSG_00477GzC2H003 NoneReduced ReducedNormal
FGSG_10517GzC2H090 None ReducedReduced Reduced
FGSG_13746 GzNot002None Reduced ReducedIncreased
FGSG_10129FgStuANoneReducedReducedNone
FGSG_10384GzAPSES004None ReducedReducedNone
FGSG_06291GzBrom002NoneReducedReducedNone
FGSG_05171GzbZIP007 NoneReducedNo growthNone
FGSG_13711GzC2H105None ReducedNo growthNone
FGSG_10716GzCCHC011None ReducedNo growthNone
FGSG_01665FgFSR1None ReducedReducedNone
FGSG_09992GzNH001 NoneReducedReducedNone
FGSG_13120 GzOB047None ReducedReducedNone
FGSG_06948GzscpNone ReducedReducedNone
FGSG_08481GzWing018None ReducedReducedNone
FGSG_08572GzWing019None Reduced ReducedNone
FGSG_08719GzWing020None ReducedReducedNone
FGSG_08769GzZC108 NoneReduced ReducedNone
FGSG_07067 GzZC232None ReducedReducedNormal
FGSG_06071 GzAT001None ReducedReducedNormal
FGSG_01350 GzC2H014NoneReduced ReducedReduced
FGSG_06871GzC2H045 None ReducedReducedNormal
FGSG_00324GzMyb002 NoneReduced ReducedReduced
FGSG_10069GzZC087 NoneReduced ReducedNormal
FGSG_00574 GzZC302None Reduced ReducedNormal
FGSG_04220GzAPSES001 None ReducedReduced Reduced
FGSG_01022 GzC2H007 NoneReduced NormalReduced
FGSG_04134GzCON7None ReducedNo growthReduced
FGSG_05304GzCCAAT004None ReducedReduced Reduced
FGSG_00385 GzHMG002None ReducedReduced Reduced
FGSG_09339GzMADS003None ReducedNormalReduced
FGSG_12781 GzMyb017None ReducedReducedReduced
MM, minimal medium; DON, deoxynivalenol. Two TFs (FgStuA and FgFSR1) were characterized in the previous studies [19,21].
doi:10.1371/journal.ppat.1002310.t001
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belonging to Group 5 still made initial structures of asci or rosettes
(Figure S4). Five of the six mutants with normal perithecia
development produced abnormally-shaped ascospores (Group 6),
and one mutant produced neither asci nor ascospores (Group 7)
(Table 2 and Figure 3). In total, 96 mutants had defects in
perithecia development, five mutants exhibited defects in asco-
spore production with normal perithecia development, and four
mutants showed enhanced sexual reproduction.
Relationship between phenotype, TF conservation, and
TF expression level
We utilized BLASTMatrix tool that is available on the
Comparative Fungal Genomics Platform (CFGP, http://cfgp.
riceblast.snu.ac.kr/) [26] to investigate whether TFs whose
deletion resulted in a mutant phenotype are conserved in other
fungal species. The distribution of each F. graminearum TF in other
fungal species was surveyed using BLAST search scores. Total
BLAST scores across the 164 species (159 fungi and five
oomycetes) were obtained (Table S9). Higher scores for TFs
imply that it is more distributed across fungal species. We divided
the TF deletion mutants showing mutant phenotypes into
subgroups based on the number of mutant phenotypes as well as
phenotypic categories. The TFs whose mutants exhibited mutant
phenotypes have higher BLAST scores than those TFs without
mutant phenotypes (p=1.01610211). In particular, TF mutants
with more than six mutant phenotypes have significantly higher
BLAST scores than those of TF mutants without a mutant
phenotype (p,0.05, Fisher LSD test, Figure 4A and Table S10).
Loss of TFs unique to F. graminearum showed mutant phenotypes in
only 6% of the resulting mutants (2/31) compared to 26% of total
tested F. graminearum TFs, and their expression levels were
relatively low compared to the other 626 F. graminearum TFs.
These unique TFs may function under specific conditions which
we may not have tested in this study (Table S10).
Phenotypic analysis revealed that perithecia development was
identified as central among the tested phenotypes. Thus, we
examined whether TFs that show perithecia development defects
when deleted are widely distributed in other fungal species.
Interestingly, TFs whose deletion results in no perithecia
production are more widely distributed in other fungal species
than those of TF whose loss results in no phenotype change
(p,0.01, Duncan multiple range test, Figure 4B). Furthermore,
TFs whose deletion results in no perithecia production showed
significantly higher BLAST scores than those of TF whose loss
results in normal perithecia production (p,0.05, Duncan’s
multiple range test, Figure 4D).
The relationship between function and gene expression of F.
graminearum TFs was examined through comparison of the
phenotype dataset with the normalized spot intensity of F.
graminearum. We downloaded 121 slides from pLEXdb (http://
www.plexdb.org/) and normalized spot intensity to measure the
expression level of each gene. The normalized spot intensity of
TFs was calculated for each slide and then an average intensity
was determined as the level of gene expression (Table S11). The
TF mutants with mutant phenotypes had higher levels of gene
expression than those of TF whose deletion results in no mutant
phenotype (p=1.6061027). Mutant phenotype TFs showed
higher levels of gene expression than those of TFs whose deletion
results in no mutant phenotype, and mutant phenotype TFs with
no perithecia production exhibited higher levels of gene expression
than those of mutant phenotype TFs with normal perithecia
production (p,0.01, Duncan’s multiple range test, Figure 4C).
However, no different expression patterns were observed among
the TFs with mutant phenotypes in sexual development even in
perithecia development-related microarrays (Figure S8) [5,6].
Overall, phenotype and expression level of TFs as well as
phenotype and distribution of TFs in other fungal species is highly
correlated with F. graminearum TFs.
Interconnection of mutant phenotype TFs through
phenotype, expression, and sequence
Dissecting transcriptional regulatory networks (TRNs) and
determining which TFs participate in individual cellular processes
and how they interact are main goals in the field of TF research.
Analyses of mutants showing mutant phenotypes provide valuable
information for addressing these questions. We attempted to
identify a TRN in F. graminearum via construction of TF
connections through patterns of gene expression and predicted
protein-protein interaction. In order to analyze TF gene
expression, we built F. graminearum gene transcript co-expression
networks for the putative TFs based on PCC, calculated using the
publicly available Affymetrix microarray data (Table S11). The
use of PCC was successfully applied previously to validate other
biological data [27]. We defined correlated and anticorrelated
interactions through values of PCC (PCC.|0.75|) that is
corresponding to approximately 0.6% portion of all PCC pairs
(Table S12). We identified 426 TF pairs that have PCC.|0.75|
(Table S12 and Figure 5A). Next, we predicted protein-protein
interaction (PPI) among TF pairs using interologs in S. cerevisiae
from the PPI database. Using this approach, 243 PPI pairs were
predicted for F. graminearum TFs (Table S13 and Figure 5B). These
co-expression and predicted PPI data were incorporated into our
phenome dataset containing 170 TFs whose deletion results in at
least one mutant phenotype (Figure 5C). Deletion of 35 out of 426
co-expressed TF pairs and 25 out of 243 predicted PPI pairs
resulted in a mutant phenotype (Figure 5D). These interconnected
48 TFs have higher BLAST scores (p=2.2361024) than the
remaining 122 TFs whose deletion results in a mutant phenotype.
Thus, these interconnected TFs, which are members of various
subfamilies, may coregulate diverse functions.
Discussion
Taking advantage of genome sequences of several model
organisms, genome-wide high-throughput genetic screens have
been performed through the use of gene disruption methods
[28,29] or RNA interference [30,31]. For fungi, the construction
Table 2. Classification of transcription factors (TFs) that are
crucial for sexual development.
Group
Perithecia
development
Ascospore
formation Number
1Increased number Normal4
2 NoneNone44
3Defective Normal23
4Defective Abnormal shaped9
5DefectiveNo ascospore 19
6 Normal Abnormal shaped5
7 NormalNo ascospore1
Groups were classified based on perithecia development (number or
maturation) and ascospore formation (existence and morphology). ‘‘Defective’’
in perithecia development means decreased number or defective maturation.
‘‘Number’’ represents the number of TFs included in each group.
doi:10.1371/journal.ppat.1002310.t002
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