Genome Biology 2008, 9:R85
2008 Ohet al. Volume 9, Issue 5, Article R85
Transcriptome analysis reveals new insight into appressorium
formation and function in the rice blast fungus Magnaporthe oryzae
YeonyeeOh*, NicoleDonofrio*‡, Huaqin Pan*§, Sean Coughlan†,
Douglas E Brown*, Shaowu Meng*, Thomas Mitchell*¶ and Ralph A Dean*
Addresses: *North Carolina State University, Center for Integrated Fungal Research, Raleigh, NC 27695-7251, USA. †Agilent Technologies,
Little Falls, DE 19808-1644, USA. ‡Current address: University of Delaware, Department of Plant and Soil Science, Newark, DE 19716, USA.
§Current address: RTI international, Research Triangle Park, NC 27709-2194, USA. ¶Current address: Ohio State University, Department of
Plant Pathology, Columbus, OH 43210, USA.
Correspondence: Ralph A Dean. Email: ralph_ email@example.com
© 2008 Oh et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Magnaporthe oryzae appressonium formulation<p>Analysis of genome-wide gene-expression changes during spore germination and appressorium formation in <it>Magnaporthe oryzae</it> revealed that protein degradation and amino-acid metabolism are essential for appressorium formation and subsequent infec-tion.</p>
Background: Rice blast disease is caused by the filamentous Ascomycetous fungus Magnaporthe
oryzae and results in significant annual rice yield losses worldwide. Infection by this and many other
fungal plant pathogens requires the development of a specialized infection cell called an
appressorium. The molecular processes regulating appressorium formation are incompletely
Results: We analyzed genome-wide gene expression changes during spore germination and
appressorium formation on a hydrophobic surface compared to induction by cAMP. During spore
germination, 2,154 (approximately 21%) genes showed differential expression, with the majority
being up-regulated. During appressorium formation, 357 genes were differentially expressed in
response to both stimuli. These genes, which we refer to as appressorium consensus genes, were
functionally grouped into Gene Ontology categories. Overall, we found a significant decrease in
expression of genes involved in protein synthesis. Conversely, expression of genes associated with
protein and amino acid degradation, lipid metabolism, secondary metabolism and cellular
transportation exhibited a dramatic increase. We functionally characterized several differentially
regulated genes, including a subtilisin protease (SPM1) and a NAD specific glutamate
dehydrogenase (Mgd1), by targeted gene disruption. These studies revealed hitherto unknown
findings that protein degradation and amino acid metabolism are essential for appressorium
formation and subsequent infection.
Conclusion: We present the first comprehensive genome-wide transcript profile study and
functional analysis of infection structure formation by a fungal plant pathogen. Our data provide
novel insight into the underlying molecular mechanisms that will directly benefit efforts to identify
fungal pathogenicity factors and aid the development of new disease management strategies.
Published: 20 May 2008
Genome Biology 2008, 9:R85 (doi:10.1186/gb-2008-9-5-r85)
Received: 21 December 2007
Revised: 18 March 2008
Accepted: 20 May 2008
The electronic version of this article is the complete one and can be
found online at http://genomebiology.com/content/9/5/R85
Genome Biology 2008, 9:R85
Genome Biology 2008, Volume 9, Issue 5, Article R85 Oh et al. R85.2
In the course of evolution, organisms have adapted to exploit
diverse habitats, including the ability to grow and reproduce
at the expense of others. Many pathogens have evolved
sophisticated strategies to first attach to and subsequently
infect their hosts, processes that often involve unique mor-
phological changes. Discovery of the underlying molecular
mechanisms of how pathogens first recognize hosts and set in
motion the infection process is not only central to under-
standing pathogen biology, but requisite for the development
of effective disease control strategies. The perception of cues
from a host typically trigger a cascade of cellular processes
whereby a signal is relayed from the cell surface to the
nucleus, resulting in activation of gene expression and, in the
case of many fungal pathogens, specific developmental
changes. Magnaporthe oryzae is typical of many fungal path-
ogens of plants in that it elaborates a specialized infection cell
called an appressorium to infect its host. M. oryzae is the
causal agent of rice blast, the most destructive fungal disease
of rice worldwide and a seminal model for the study of the
molecular basis of fungal-plant interactions. It was the first
filamentous fungal pathogen to have a complete genome
sequence publicly available .
Following spore attachment and germination on the host sur-
face, an emerging germ tube perceives physical cues, such as
surface hardness and hydrophobicity, as well as chemical sig-
nals, including wax monomers, that trigger appressorium for-
mation [2-4]. Appressorium formation begins when the tip of
the germ tube ceases polar growth, hooks, and begins to swell.
The contents of the spore are then mobilized into the develop-
ing appressorium, a septum develops at the neck of the
appressorium, and the germ tube and spore collapse and die.
As the appressorium matures, it becomes firmly attached to
the plant surface and a dense layer of melanin is laid down in
the appressorium wall, except across a pore at the plant inter-
face. Turgor pressure increases inside the appressorium and
a penetration hyphae emerges at the pore, which is driven
through the plant cuticle into the underlying epidermal cells
[5-10]. Melanin deposition in the cell wall of the appresso-
rium is essential for maintaining turgor pressure. Genetic
mutations or chemical treatments that inhibit appressorium
formation and function effectively block penetration and sub-
sequent disease development [7,11].
Highly conserved signaling networks that transfer cues from
the environment to the nucleus play a crucial role in regulat-
ing pathogen-host interactions. For M. oryzae, the mitogen-
activated protein kinase (MAPK), cyclic AMP (cAMP) and to
a lesser extent Ca2+ signaling pathways have been shown to be
essential for appressorium formation and function [12-16]. In
addition, the cAMP signaling pathway regulates several other
aspects of fungal growth and development, including nutrient
sensing and cell morphogenesis [17-19]. In M. oryzae, exoge-
nous cAMP and analogs induce appressorium formation in
non-inductive environments . Subsequent functional
characterization of genes encoding proteins in the cAMP sig-
naling pathway, including MagB, alpha subunit of G protein,
Mac1, adenylyl cyclase, and cPKA, the catalytic subunit of
protein kinase A, provided clear evidence for the essential
role of cAMP in regulating appressorium morphogenesis
[13,21-23]. These pioneering studies served as the catalyst to
drive numerous studies in other pathogenic fungi such as
Blumeria, Colletotricum, Fusarium, and Sclerotinia species
[24-27]. However, while the core pathways are highly con-
served, relatively little is known of the downstream genes and
pathways that direct infection related morphogenesis.
Appressorium function is dependent on generating high lev-
els of turgor, which in M. oryzae results from high concentra-
tions of glycerol. How glycerol is generated in the appressoria
remains to be clearly defined, but because appressoria
develop in the absence of nutrients, it has been suggested that
glycerol must be derived from storage products. Carbohy-
drate catabolism in yeast is regulated by the cAMP response
pathway; however, there is no genetic evidence that metabo-
lism of storage glycogen or trehalose is required for appresso-
rium turgor generation . TRE1, which encodes the main
intracellular trehalase activity in spores, is not required for
appressorium function . On the other hand, targeted
mutagenesis of genes involved in degradation of storage lip-
ids or beta oxidation of fatty acids, such as MFP1, or genes
involved in peroxisome function, such as MgPEX6, prevent
appressorium function but do not appear to affect the accu-
mulation of glycerol . Thus, although spores and develop-
ing appressoria contain substantial amounts of lipids and
carbohydrates, it appears that glycerol may be derived from
other cellular materials. Appressorium formation is accom-
panied by collapse of the spore, a process involving autophagy
whereby cellular contents of the spore are re-cycled into the
developing appressorium. Autophagy genes MgATG1 or
MgATG8 are required for normal appressorium formation
and deletion mutants are non-pathogenic [31,32]. This opens
up the possibility that glycerol may be derived from materials
other than lipids and carbohydrates.
Studies of appressorium formation and early stages of host
invasion suggest that M. oryzae is not only capable of perceiv-
ing its host but is able to evade host detection during pre-pen-
etration and tissue colonization . Bacteria have evolved a
specialized type III secretion system to deliver proteins into
plant cells to help evade host recognition and promote inva-
sive growth . M. oryzae mutants defective in secretion,
MgAPT2 deletion strains, for example, are unable to cause
disease . Thus, secreted proteins likely play a significant
role in fungal pathogenesis. The M. oryzae genome contains
a large and diverse complement of secreted proteins; how-
ever, their function remains largely unknown. Other effector
molecules, including secondary metabolites, may be deliv-
ered by transporters. It is known that ATP-binding cassette
(ABC) type transporters such as ABC3 are required for
appressorium function . M. oryzae contains at least 23
Genome Biology 2008, Volume 9, Issue 5, Article R85 Oh et al. R85.3
Genome Biology 2008, 9:R85
polyketide synthases, several non-ribosomal peptide syn-
thases and more than 120 highly diverged cytochrome P450
monooxygenases, suggesting a significant capacity to produce
a diverse array of secondary metabolites . The nature of
these metabolites and the role they play in the infection proc-
ess is not well defined.
Although evidence collected to date, primarily from studies of
M. oryzae, provides important clues as to processes involved
in appressorium formation and function, a complete under-
standing of the metabolic changes and genes contributing to
infection related morphogenesis is far from complete. One
powerful method for refining and extending knowledge of the
infection process is to identify alterations in transcription as
M. oryzae undergoes appressorium formation. To date, very
limited gene expression studies have been performed to iden-
tify genes associated with appressorium formation and func-
tion in fungal pathogens [37-43]. Published studies have
examined only small subsets of the total gene complement
from fungal pathogens and have been far from exhaustive.
The recent completion of the M. oryzae genome sequence
greatly enables genomic analyses .
In this study, we made use of a whole genome oligo micro-
array chip containing over 13,000 M. oryzae elements repre-
senting 10,176 predicted genes, and conducted global gene
expression profiles during spore germination and appresso-
rium formation on both an inductive hydrophobic surface
and in response to cAMP (Figure 1). From these data, we dis-
tilled a consensus set of genes differentially expressed in
response to both physical and chemical cues, and constructed
putative biological pathways that participate in appressorium
formation. Our data show that germination stimulates a
major transcriptional response characterized by a dramatic
increase in expression of genes involved in metabolism and
biosynthesis. On the other hand, induction of appressorium
formation triggers a significant decrease in expression of
genes associated with the translational apparatus, with a
coordinate increase in the expression of genes involved in
protein and amino acid degradation, lipid metabolism, sec-
ondary metabolism and cellular transportation. Significantly,
the set of up-regulated genes is enriched for those encoding
predicted secreted proteins. To directly assay the role of these
gene sets in appressorium formation and function, we per-
formed targeted gene deletion studies on many of the most
highly up-regulated genes. Our findings reveal that protein
degradation and amino acid metabolism are essential for the
infection process. Further, we find many differentially
expressed genes are not required for appressorium formation
and function. This may suggest that M. oryzae employs a
number of backup systems, such as functional redundancy
and compensatory processes in order to protect appresso-
rium formation from being de-regulated.
Genes involved in core biological processes undergo
dramatic transcriptional changes during spore
Microarray analysis revealed that about 29% of the 10,176 M.
oryzae genes present on the array underwent significant
changes (≥ 2-fold, p < 0.05) in expression during at least one
of the developmental processes tested, including spore germi-
nation, germ tube elongation or appressorium development
(Table 1). The most dramatic change in gene expression
occurred during spore germination (Phil7 versus Spore)
where approximately 21% showed differential expression
with the vast majority being up-regulated. Seventy three per-
cent of the genes differentially expressed during spore germi-
nation exhibited no further change in expression during germ
tube elongation or appressorium formation. Very few further
changes (<1%) in gene expression were observed during germ
tube elongation (Phil12 versus Phil7).
To explore the cellular processes active during spore germi-
nation, differentially expressed genes were first grouped
according to Gene Ontology (GO) terms. Examination of gene
expression with GO categories revealed that during spore ger-
mination, genes involved in major biological processes such
as metabolism (GO:0008152)
(GO:0009058) were significantly over-represented (p < 0.01)
in the up-regulated gene set (Additional data file 1). In partic-
ular, genes associated with carbohydrate metabolism
(GO:0005975), amino acid and derivative metabolism
(GO:0006519) and protein metabolism (GO:0019538) were
over-represented. In contrast, genes associated with the GO
category for transcription (GO:0006350) were under-repre-
sented in the up-regulated gene set. The GO category for tran-
scription contains mainly transcription factors and other
proteins involved in DNA binding. Typically, transcription
factors are post transcriptionally regulated and, thus, their
expression would not necessarily be expected to be over-rep-
resented during spore germination (Additional data file 1).
Thigmotrophic and chemical induction of
appressorium formation trigger similar patterns of
Approximately 3-4% of the entire set of M. oryzae open read-
ing frames were differentially expressed during appressorium
initiation (Pho7 versus Phil7) and maturation (Pho12 versus
Phil12) on the inductive surface. In response to exogenous
cAMP, about 10% of expressed genes were differentially
expressed (cAMP9 versus Phil9; Table 1). Considerably more
genes were found to be induced rather than repressed by both
physical and chemical (cAMP) stimulation. Overall, good cor-
relations (Pearson's correlation coefficient r > 0.5) were
observed between appressorium related expression profiles
induced by physical cues (appressorium initiation and matu-
ration) and by cAMP (Figure 2). In contrast, gene expression
profiles during spore germination and germ tube elongation
correlated poorly with those observed for appressorium
Genome Biology 2008, 9:R85
Genome Biology 2008, Volume 9, Issue 5, Article R85 Oh et al. R85.4
Figure 1 (see legend on next page)
Incubated for 7
and 12 hours
+ cAMP (50mM)
Full loop design
Incubated for 7
and 12 hours
App. non inductive
App. non inductive
Incubated for 9
App. non inductive
Incubated for 9
Genome Biology 2008, Volume 9, Issue 5, Article R85 Oh et al. R85.5
Genome Biology 2008, 9:R85
formation. The highest correlation (r = 0.66) was found
between appressorium maturation and cAMP induced
appressoria where 66% of differentially expressed genes
showed a similar expression pattern. Approximately 54% of
genes differentially expressed during appressorium initiation
exhibited a similar expression pattern in response to cAMP
(Table 1, Figure 2).
Microarray based gene expression pattern is consistent
with expression analysis from reverse transcriptase
PCR and quantitative RT-PCR
To confirm gene expression patterns derived from our micro-
array experiments, we performed reverse transcriptase PCR
(RT-PCR) with five selected up-regulated genes, three down-
regulated, and two showing no expression change (Figure 3).
Genes were selected based on their overall expression levels,
that is, represented high to medium to low expressed genes. If
genes contain an intron, primers were designed to bridge the
intron to distinguish amplification of transcript from any pos-
sible genomic DNA contamination (Additional data file 2).
RT-PCR results were consistent with the microarray data,
albeit the absolute levels of expression fold change showed
slight variation (Figure 3). Two genes, MPG1 and PTH11
[44,45], were also subjected to analysis by quantitative RT-
PCR (qRT-PCR). Both genes are required for pathogenesis.
MPG1 has been shown to be highly expressed during appres-
sorium formation . However, our microarray and RT-
PCR results indicated that both genes were more strongly up-
regulated during germ tube elongation than appressorium
formation (Figure 3).
Appressorium consensus gene sets reveal key
biological processes for appressorium formation
To identify genes that participate in appressorium formation,
we compared gene expression profiles of appressorium initi-
ation, maturation and cAMP induced appressoria. A total of
240 genes were up-regulated and 117 were down-regulated
during appressorium initiation or maturation and in
response to cAMP (Figure 4). These genes, referred to as
appressorium consensus genes, were functionally grouped
into GO categories based on manual curation as described in
Materials and methods (Figure 4 and Additional data file 3).
Overall, we noted a significant decrease in expression of genes
involved in protein synthesis during appressorium induction.
On the other hand, expression of genes associated with pro-
tein and amino acid degradation, lipid degradation, second-
ary metabolism, including melanin biosynthesis, and cellular
transportation exhibited a dramatic upshift. Moreover, this
set of genes exhibited nearly a four-fold enrichment for genes
encoding secreted proteins. A detailed discussion of the func-
tional groups exhibiting differential expression is presented
Experimental and microarray design for spore germination and appressorium induction
Figure 1 (see previous page)
Experimental and microarray design for spore germination and appressorium induction. (a) Spores were placed on the hydrophilic (Phil) and hydrophobic
(Pho) surfaces of GelBond and incubated for 7 and 12 h. For induction of appressoria by cAMP, spores were placed on the hydrophilic surface of GelBond
with (cAMP9) and without (Phil9) cAMP and incubated for 9 h. (b) Diagrams show microarray design. Arrows connect samples directly compared on two
channel Agilent M. oryzae oligonucleotide microarrays. Arrow heads = Cy5, arrow tails = Cy3.
Differential expression of 10,176 M. oryzae genes during spore germination and appressorium formation
SG_up* SG_dnGE_upGE_dn AI_upAI_dn AM_up AM_dnCI_up CI_dn
*Abbreviations indicate particular expression profiles: SG, spore germination; GE, germ tube elongation; AI, appressorium initiation; AM,
appressorium maturation; CI, cAMP induced appressorium. Up, up-regulated; dn, down-regulated. †Each value indicates the number of genes in
common between the paired expression profiles.
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Genome Biology 2008, Volume 9, Issue 5, Article R85 Oh et al. R85.6
Major changes in amino acid and protein metabolism
Two of the major functional categories of genes in the up-reg-
ulated appressorium consensus gene set were those with pre-
dicted roles in protein and amino acid degradation. Protein
sequence analysis of putative proteases recognized subgroups
according to the active site and substrate specificity, such as
acid proteases (MGG_ 03056.5, MGG_ 09032.5), aspartyl
proteases (MGG_ 09351.5, MGG_ 00981.5), subtilisin-like
proteases (MGG_ 03670.5, MGG_ 09246.5), calpain (cal-
proteases (MGG_ 08526.5, MGG_ 03260.5), a cysteine pro-
tease required for autophagy (MGG_ 03580.5), a carboxy-
lpeptidase (MGG_ 09716.5), and a tripeptidyl peptidase
MGG_ 03670.5 (named SPM1) and MGG_ 09246.5 are puta-
tive proteases bearing the signature for subtilisin peptidase. A
BLASTp search revealed that SPM1 and MGG_ 09246.5 have
39% amino acid identity and 55% similarity to each other and
both match serine proteases from various microorganisms.
The possibility of SPM1 as a pathogenicity candidate in M.
oryzae was first proposed based on its prevalence in a cDNA
library of mature appressoria . SPM1 was also found to be
abundant in SAGE tags derived from cAMP induced mature
appressoria . Although SPM1 contains a predicted signal
peptide, the protein appears to be targeted to the vacuole
. As previously reported , SPM1 targeted deletion
mutants produced melanized appressoria but exhibited
severely reduced pathogenicity on rice and barley plants. Dis-
ease lesions failed to expand and sporulation was severely
reduced . In addition, further characterization here
revealed vegetative growth, particularly aerial hyphae, of
deletion mutants was decreased on the various nutrient
sources such as oatmeal, V8 and minimal media but little dif-
ference was observed on complete media (Figure 5). On the
other hand, targeted deletion mutants (see Materials and
methods for details) of the putative protease encoded by
MGG_ 09246.5 appeared normal and formed typical pig-
mented appressoria and developed disease symptoms on bar-
ley plants indistinguishable from wild type (Figure 6).
Protein degradation is highly regulated in many instances.
Many short-lived proteins destined for degradation are selec-
tively tagged by ubiquitin. It is noteworthy that several pro-
teins involved in this process, including polyubiquitin
(MGG_ 01282.5) and ubiquitin activating enzyme E1 like pro-
tein (MGG_ 07297.5), were up-regulated. Additionally, gene
expression of putative
(MGG_ 11888.5, MGG_ 01115.5) exhibited increased expres-
sion in response to cAMP. Following selective tagging, pro-
teins are degraded by the proteasome. Several probable 26S
proteasome regulatory protein subunits (MGG_ 05477.5,
MGG_ 05991.5, MGG_ 01581.5, MGG_ 07031.5) were up-reg-
ulated by cAMP. Currently, it is unknown which proteins are
selectively tagged or how the proteasome regulatory proteins
influence appressorium formation.
ubiquitin protein ligases
Gene expression profile clustering and correlation analysis
Gene expression profile clustering and correlation analysis. (a)
Hierarchical clustering analysis of gene expression profiless for spore
germination (SG), germ tube elongation (GE), appressorium initiation (AI),
appressorium maturation (AM) and cAMP-induced appressoria (CI).
Differential expression of each gene is indicated in color (red shows
induced, green shows repressed, and numbers next to scale indicate fold
change (log2)). (b) Correlation coefficient for pairwise gene expression
profiles shown in (a).
SG GE AI AM CI
H i erarchi cal C l ust eri ng
RT-PCR and qRT-PCR analysis of gene expression
Figure 3 (see following page)
RT-PCR and qRT-PCR analysis of gene expression. (a) RT-PCR using RNA isolated from spores germinated on the hydrophobic (Pho12) and hydrophilic
(Phil12) surfaces of GelBond after 12 h incubation compared to expression fold change (FC) derived from microarray data from the same time point. (b)
qRT-PCR analysis of MPG1 and PTH11 using RNA from appressoria induced by cAMP (+cAMP) and germinating spores (-cAMP) after 9 h incubation on
the hydrophilic surface of GelBond. Gene expression fold changes for MPG1 and PTH11 were 0.2 and 0.2, respectively, in our cAMP microarray study.
Genome Biology 2008, Volume 9, Issue 5, Article R85 Oh et al. R85.7
Genome Biology 2008, 9:R85
Figure 3 (see legend on previous page)
Pho12 Phil12 FC Gene Description
MAS3 homolog [Magnaporthe oryzae] MGG_09875.50.2
Catalase -1 [Neurospora crassa]
Actin MGG_03982.5 1.0
Predicted proteinMGG_06456.5 1.0
Laccase[Aspergillus nidulans] MGG_08523.55.3
Subtilisin -like proteinase Spm1 precursorMGG_03670.5 3.2
NAD(+)-specific glutamate dehydrogenaseMGG_05247.53.0
MPG1 gene expression
Fold change over actin level
Pth11 gene expression
Fold change over actin level
Genome Biology 2008, 9:R85
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Figure 4 (see legend on next page)
AM AMAI AI
2525 2546 4646
Cell cycle and development
Response to stress
Cell wall and surface
Electron transport and energy metabolism
Glycan biosynthesis and metabolism
Amino acid metabolism
Proteolysis and peptidolysis
Number of genes in each functional category
Genome Biology 2008, Volume 9, Issue 5, Article R85 Oh et al. R85.9
Genome Biology 2008, 9:R85
In addition to evidence for elevated protein degradation dur-
ing appressorium formation, expression of genes involved in
amino acid metabolism was also up-regulated. A putative
aminotransferase (MGG_ 09919.5), a cystathionine γ-lyase
(MG10380.5) that catalyzes the transition of cystathionine to
cysteine, 2-oxobutanoate and ammonia, a cysteine dioxygen-
ase (MG6095.5) in the cysteine degradation pathway, and a
threonine deaminase (MGG_ 07224.5) required for threonine
degradation were up-regulated. 2-Oxobutanoate (α-ketobu-
tyrate), produced by cystathionine γ-lyase and threonine
deaminase, can then be further metabolized through the
tricarboxylic acid cycle. Turnover of the cellular storage
amino acids, arginine and proline, to glutamate depends on
the nutrient status of the cell. Genes involved in arginine and
proline degradation to glutamate, such as arginase
(MGG_ 10533.5), ornithine
(MGG_ 06392.5), delta-1-pyrroline-5-carboxylate dehydro-
genase (MGG_ 00189.5),
(MGG_ 04244.5), were up-regulated during appressorium
and proline oxidase
NAD(+) dependent glutamate dehydrogenase (NAD-GDH)
provides a major conduit for feeding carbon from amino acids
back into the tricarboxylic acid cycle. The enzyme catalyzes
the oxidative deamination of glutamate to produce α-ketogl-
utarate and ammonia (glutamate + NAD+ → α-ketoglutarate
+ NH4 + NADH). Our gene expression data showed that the
M. oryzae NAD-GDH homolog MGG_ 05247.5, which we
have named Mgd1, was present in the up-regulated appresso-
rium consensus gene set. Previous work using serial analysis
of gene expression (SAGE) had shown that transcripts of
Mgd1 were abundant in mature appressoria of M. oryzae
induced by cAMP .
To evaluate the function of Mgd1 in M. oryzae, we generated
four independent targeted deletion mutants. Mutants lacked
aerial hyphae when grown on complete media (Figure 7). In
addition, growth was severely reduced on poor carbon
sources such as Tween 20 and polyethylene glycol compared
to ectopic and wild-type strains. The mutants also grow more
poorly than ectopic and wild-type strains on glucose limiting
conditions in the presence of glutamate and glutamine. NAD-
GDH gene deletion mutants in yeast and Aspergillus nidu-
lans also showed poor growth on glutamate as a sole nitrogen
source [49,50]. To determine the role of Mgd1 in virulence,
we evaluated mutants for appressorium formation and the
ability to cause disease. Mutants had a reduced ability to form
mature appressoria (45%) on an inductive surface; other
appressoria appeared immature (41%) or were abnormal and
highly swollen (4%) (Figure 6). When inoculated onto suscep-
tible barley plants, the mutants exhibited highly reduced vir-
ulence and produced many fewer and smaller lesions (Figure
6). Thus, Mgd1 appears to be required for efficient metabo-
lism of carbon and/or nitrogen from the break down of pro-
teins under nutrient limiting conditions as experienced when
cells are attempting to form appressoria.
In contrast to the activated expression of genes involved in
protein and amino acid degradation, a major portion of the
down-regulated genes encode components of the ribosome;
16 constitute the large ribosomal large subunit and 6 the
small subunit (Figure 4 and Additional data file 3). Expres-
sion of all of these genes was up-regulated during spore ger-
mination and remained unchanged during germ tube
elongation. However, upon appressorium induction the aver-
age level of expression fell 30% during appressorium initia-
tion, and by more than 2-fold during appressorium
maturation. Similar changes in gene expression patterns were
observed in appressoria induction by cAMP.
Increased gene expression for lipid metabolism
Lipids are essential components of living cells as well as
major sources of energy reserves. Several genes involved in
the synthesis of the cell membrane components were induced
during appressorium formation and they included a 7-dehy-
drocholesterol reductase (MGG_ 03765) that catalyzes the
last step of cholesterol biosynthesis pathway, oxysterol bind-
ing protein (MGG_ 00853.5) involved in cholesterol biosyn-
thesis, a probable sterol carrier protein (MGG_ 02409.5)
Functional categorization of appressorium consensus genes
Figure 4 (see previous page)
Functional categorization of appressorium consensus genes. (a) Appressorium associated expression profiles were combined and 240 up-regulated and
117 down-regulated genes were designated as appressorium consensus genes (in italics). Abbreviations are same as in Figure 2. (b) Up-regulated (in pink)
and down-regulated (in blue) genes were grouped according to their putative function.
Vegetative growth of the SPM1 deletion mutant on various nutrient sources
Vegetative growth of the SPM1 deletion mutant on various nutrient
sources. SPM1 deletion mutant (Δspm1), ectopic strain and wild type 70-
15 (WT) were incubated on solidified complete media (CM), oatmeal
media (OA), V8 media (V8) and minimal media (MM) for seven days.
Results shown are typical for all four independent SPM1 deletion mutants.
CM V8 OA MM
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Genome Biology 2008, Volume 9, Issue 5, Article R85 Oh et al. R85.10
involved in cholesterol trafficking and metabolism, glycerol-
3-phosphate acyltransferase (MGG_ 11040.5) required for
phospholipid biosynthesis, diacylglycerol cholinephospho-
transferase (MGG_ 03690.5) involved in phosphatidylcholine
biosynthesis pathway, and delta 8 sphingolipid desaturase
(MGG_ 03567.5) involved in sphingolipid metabolism. Con-
Appressorium formation and pathogenicity of targeted gene deletion mutants
Appressorium formation and pathogenicity of targeted gene deletion mutants.
Target gene Description AIAMCI
11.0 5.0 26.0
MGG_08523.5Laccase [Emericella nidulans ]
Subtilisin-like proteinase Spm1
NAD-specific glutamate dehydrogenase,
Mgd1 [Magnaporthe oryzae]
2.6 3.0 4.1
1.1 2.1 39.4
Transcription activator CRG1
12.0 5.5 4.4
Oleate-induced peroxisomal protein POX18
a appressorium development on a hydropholic surface after 20 h incubation.
b pathogenicity of target gene deletion mutants on barley seedings 5 days after inoculation.
c numbers indicate gene expression fold change for appressorium induction (AI), appressorium maturation (AM)
and cAMP induced appressoria (CI).
Genome Biology 2008, Volume 9, Issue 5, Article R85 Oh et al. R85.11
Genome Biology 2008, 9:R85
Figure 7 (see legend on next page)
Mgd1a ectopic a Mgd1a ectopic a
ectopic b WT ectopic b WT
Genome Biology 2008, 9:R85
Genome Biology 2008, Volume 9, Issue 5, Article R85 Oh et al. R85.12
versely, gene expression of fatty acid omega hydroxylases
(MGG_ 10879.5, MGG_ 01925.5) and a cholinesterase
(MGG_ 02610.5) involved in lipid degradation was found to
Beta-oxidation of fatty acids in fungi occurs mainly in the per-
oxisome. Peroxisomes are membrane bound subcellular
organelles where diverse anabolic and catabolic metabolisms,
including peroxide metabolism, glyoxylate metabolism, and
phospholipid biosynthesis, are conducted . During fatty
acid metabolism, the very long chain fatty acids (C22 or
longer) are first transferred to coenzyme A by a very long
chain fatty acyl-CoA synthetase. In Saccharomyces cerevi-
siae, very long chain fatty acyl-CoA synthetase, FAT1, disrup-
tion mutants showed reduced growth on media containing
dextrose and oleic acid and very long chain fatty acids
accumulated in cells . Our data showed that a very long
chain fatty acyl-CoA synthetase (MGG_ 08257.5) was up-reg-
ulated, suggesting fatty acid catabolism is involved in appres-
sorium formation and function.
Recently, several genes for peroxisome structure, transloca-
tion of peroxisomal target proteins and metabolism in the
peroxisome have been shown to be involved in pathogenicity,
cellular differentiation and nutrient assimilation in fungi [53-
56]. In M. oryzae, isocitrate lyase (ICL1) of the glyoxylate
cycle, a HEX1 ortholog, PTH2 peroxisomal acetyl carnitine
transferase, the multifunctional β-oxidation protein MFP1
and MgPex6, which is required for peroxisome biogenesis,
were found to be necessary for functional appressorium
development and fungal infection [30,57-59]. A putative fatty
acid binding peroxisomal protein (MGG_ 07337.5) was
identified in the up-regulated set of appressorium consensus
genes. MGG_ 07337.5 encodes a protein with 40% identity
and 59% similarity to the peroxisomal non-specific lipid
transfer protein PXP-18, which is encoded by POX18 from
Candida tropicalis and is highly conserved in filamentous
fungi. POX18 mRNA was shown to be enriched by oleic acid
and n-alkane rather than by glucose. PXP-18 appears to func-
tion in peroxisomal production of acetyl-coA either by guid-
ing lipids through the oxidation processes or by protecting the
acyl-coA oxidase enzyme [60-63]. To address the function of
the PXP-18 homolog in M. oryzae, targeted deletion mutants
were created. However, despite other evidence for the role of
the peroxisome in appressorium formation and plant infec-
tion [30,53,64], the putative
MGG_ 07337.5 was found to be dispensable for the develop-
ment of a mature pigmented appressorium and disease symp-
tom development on barley and rice plants in this study.
Mutants were indistinguishable from wild type for other
aspects of growth and development examined (Figure 6).
Carbohydrate metabolism: cell wall degradation, remodeling and
carbon scavenging during appressorium development
Carbohydrates represent a major component of fungal bio-
mass. Glycogen and various polyols are significant storage
carbohydrates, whereas chitin, glucans and other polymers
are primary constituents of the fungal cell wall. Inspection of
our microarray gene expression analysis revealed a group of
genes encoding enzymes for cell wall degradation, glucan
mobilization and cell wall glycoprotein processing in the set
of appressorium consensus genes. Several chitinase genes,
such as MGG_ 00086.5 and MGG_ 01876.5, a beta-1,3
exoglucanase (MGG_ 00659.5),
(MG10038.5), and polysaccharide
(MGG_ 01922.5), were up-regulated. However, other genes
encoding glucan degrading enzymes showed opposite
expression profiles. For example, glucan 1,4-alpha-glucosi-
dase (MGG_ 01096.5),
(MGG_ 05364.5), beta-1,3-glucosidase (MGG_ 10400.5) and
alpha-L-fucosidase (MGG_ 00316.5) were down-regulated.
The gene expression of a putative cell wall degrading protein
(MGG_ 03307.5) containing a LysM associated with general
peptidoglycan binding function and a glucosamine-6-phos-
phate deaminase (MG10038.5) in the glucosamine degrada-
tion pathway was increased but a UDP-N-acetylglucosamine-
pyrophosphorylase (MGG_ 01320.5) that catalyzes the for-
mation of UDP-N-acetyl-D-glucosamine, which is an essen-
tial precursor of cell wall peptidoglycan lipopolysaccharide,
was repressed. These results suggest dynamic changes in glu-
can metabolism occur during appressorium formation.
Fungal cell walls contain high levels of mannoproteins (30-
50% in yeast cell walls). These proteins are first glycosylated
(glycosylphosphatidylinositol anchored) by mannosyltrans-
ferase adding mannose to their serine or threonine amino
acid residues and then further processed by mannosidases.
During appressorium development, several genes encoding
these enzymes, alpha-1,2-mannosyltransferase
(MGG_ 10494.5), alpha-1,6
(MGG_ 03361.5) and alpha-mannosidase (MGG_ 00994.5)
were up-regulated. In addition, gene expression of other
homologs of an alpha-1,6-mannosyltransferase
Vegetative growth of Mgd1 deletion mutants on various nutrient sources
Figure 7 (see previous page)
Vegetative growth of Mgd1 deletion mutants on various nutrient sources. (a-h) Wild type 70-15 (a-d) and Mgd1 deletion mutant (e-h) were grown on
minimal media (a,e), complete media (b,f), minimal media with Tween 20 (c,g) or polyethylene glycol (d,h) as carbon source for seven days. Results shown
are typical of all four independent Mgd1 deletion mutants. Results for ectopic strains were similar to wild type (data not shown). (i-k) Mgd1 deleted
mutants (ΔMgd1a, ΔMgd1b), ectopic strains (ectopic a, ectopic b) and wild type 70-15 (WT) were grown for seven days on minimal media (0.125%
glucose) with glutamine (i) and glutamic acid (j) as nitrogen source and minimal media (1% glucose) (k) as depicted in (l). Photographs in (i-k) were taken
on a light box to highlight differences in mycelial density.
Genome Biology 2008, Volume 9, Issue 5, Article R85 Oh et al. R85.13
Genome Biology 2008, 9:R85
(MGG_ 00163.5), and a mannosidase (MGG_ 00084.5) were
increased by cAMP treatment, implying that the active pro-
duction of mannoproteins might aid to stabilize the cell wall
during the rapid expansion of the appressorium. This hypo-
thesis is supported by the finding that expression of two genes
(MGG_ 03436.5 and MGG_ 02778.5), which encode putative
mannosylated proteins, was strikingly increased. Expression
of MGG_ 03436.5 was the most highly up-regulated in the
appressorium consensus gene set (56.6-, 59.6-, 76.9-fold
changes for appressorium initiation (AI), appressorium mat-
uration (AM) and cAMP-induced appressoria (CI), respec-
tively). MGG_ 03436.5 is a small hypothetical protein
composed of 169 amino acids in 3 exons with no known func-
tional domains. The deduced amino acid sequence showed
23% and 21% identity to that of A. nidulans cell wall manno-
protein MnpA (7.E-04) and Aspergillus flavus antigenic cell
wall protein MP1 (1.E-04), respectively. Interestingly, a puta-
tive paralog of MGG_ 03436.5 in M. oryzae, MGG_ 02778.5,
was also highly up-regulated in appressorium maturation and
response to cAMP (15.5- and 15.1-fold changes for AM and CI,
respectively). MGG_ 02778.5 was previously reported as the
most abundant expressed sequence tag in a subtracted
mature appressorium cDNA library . The function of
MGG_ 03436.5 was investigated by targeted mutagenesis.
Surprisingly, no noticeable phenotypic differences, including
MGG_ 03436.5 gene deleted mutants were observed (Figure
6). This may suggest functional redundancy among related
cell wall proteins. The mnpA-null mutant in A. nidulans
showed an irregular outer cell wall layer; however, no pheno-
typic differences in spore germination, growth and cellular
development were observed [65,66].
and pathogenicity, in
The expression of other genes involved in the utilization of
non-preferred carbon sources was also up-regulated during
appressorium formation. For example, coupled with
increased expression of a transcriptional activator of alcohol
metabolism (MGG_ 02129.5), an alcohol dehydrogenase I
(MGG_ 03880.5) and a NAD+-aldehyde dehydrogenase
(MGG_ 03263.5), which is involved in ethanol degradation
for the production of acetyl-CoA, were up-regulated. Like-
wise, another gene involved in sugar alcohol degradation, L-
arabinitol 4-dehydrogenase (MGG_ 01231.5), which hydro-
lyzes L-arabinitol to L-xylulose, was up-regulated. Gene
expression of rhamnosidase A (MGG_ 05246.5), galactose
oxidase (MG10878.5), a putative cytochrome P450 for alkane
assimilation (MGG_ 05908.5) and lactate dehydrogenase
(MGG_ 05735.5) was increased but expression of the dTDP-
D-glucose 4,6-dehydratase in the rhamnose biosynthesis
pathway (MGG_ 09238.5) and 2-isopropylmalate synthase in
the leucine biosynthesis pathway (MGG_ 13485.5) were
Secondary metabolism during appressorium formation
Fungi produce an extensive array of secondary metabolites
derived from a number of different biochemical pathways,
including the polyketide, isoprenoid and shikimate acid path-
ways, as well as through modification of amino acids.
Polyketides constitute a large class of secondary metabolites
produced by filamentous fungi. They are synthesized from
large multi-domain enzymes, polyketide synthases (PKSs)
that share significant similarities to fatty acid synthases.
Polyketide synthesis requires the coupling of malonyl-CoA to
the elongating chain. It is noteworthy that the expression of
MGG_ 07613.5, a putative acetyl-CoA carboxylase, the
enzyme that catalyzes carboxylation of acetyl-CoA to produce
malonyl-CoA, was up-regulated in the appressorium consen-
sus gene set.
Melanin is one of the most thoroughly studied polyketides in
M. oryzae and other pathogenic fungi. The three genes
involved in the synthesis of dihydroxynaphthalene-melanin,
a PKS, a synthalone dehydratase (SCD), and a hydroxynaph-
thalene reductase (THR), are clustered in the plant patho-
genic fungus Alternaria alternata and the opportunistic
human pathogen Aspergillus fumigatus. Targeted gene dis-
ruption experiments showed that these genes are essential for
spore pigmentation and fungal pathogenicity [67-69]. In M.
oryzae, the melanin biosynthesis genes ALB1, RSY1 and
BUF1 are required for appressorium function but are not
clustered [7,11]. ALB1, RSY1 and BUF1 correspond to
MGG_ 07219.5 (a PKS), MGG_ 05059.5 (a SCD) and
MGG_ 02252.5 (a THR), respectively. In our study,ALB1 and
RSY1 genes were present in the set of appressorium consen-
sus genes and were highly up-regulated (Additional data file
3). BUF1 was found to be up-regulated during appressorium
initiation (3.9-fold change, p = 0.056) and significantly
induced by cAMP (15.6-fold change, p < 0.001). All three
genes were most highly induced during appressorium initia-
tion, which is consistent with observations reported previ-
ously for their putative orthologs, PKS1, SCD1 and THR1, in
the anthracnose fungus Colletotrichum lagenarium [70,71].
It is noteworthy that closer inspection of the genomic region
on chromosome I containing the PKS ALB1 revealed the pres-
ence of a BUF1 homolog, MGG_ 07216.5. Positioned between
these two genes is MGG_ 07218.5, a putative transcription
factor. All three genes exhibited similar expression patterns
during appressorium formation (Additional data file 4).
MGG_ 07218.5 has an open reading frame of 1,926 nucleo-
tides, potentially encoding a protein of 487 amino acids with
a GAL4-like Zn2Cys6 binuclear cluster DNA-binding domain.
A similar domain is also found in the Pig1 transcription factor
(MGG_ 07215.5), previously reported to regulate mycelial
melanin biosynthesis in M. oryzae . Pig1 is located next to
the BUF1 homolog (MGG_ 07216.5). However, Pig1 is not
required for pathogenicity  and was not found to be dif-
ferentially expressed in our experiments.
Although it is widely considered that the PKS gene
MGG_ 07219.5 encodes ALB1, published evidence appears to
be absent. Thus, to confirm that MGG_ 07219.5 corresponds
Genome Biology 2008, 9:R85
Genome Biology 2008, Volume 9, Issue 5, Article R85 Oh et al. R85.14
to ALB1, targeted gene knock out mutants were created.
Mutants exhibited the expected albino phenotype and were
non-pathogenic on rice and barley. However, deletion of the
putative transcription factor MGG_ 07218.5 resulted in no
significant phenotypic changes in growth on various carbon
and nitrogen sources or sporulation. In addition, mutants
produced appressoria of normal appearance on a hydropho-
bic surface and produced disease symptoms on barley indis-
tinguishable from wild type (Figure 6). Thus, this
transcription factor does not appear to regulate melanin pro-
duction, at least on its own. Examination of the promoter
regions of ALB1 (MGG_ 07219.5) and the BUF1 homolog
(MGG_ 07216.5) revealed putative GAL4 type transcription
factor binding sites. GAL4 type transcription factors com-
monly form both homo- and heterodimers. Whether it is no
more than a coincidence that these two transcription factors
are physically associated with this genomic region or perhaps
together both regulate melanin biosynthesis during appresso-
rium formation awaits further investigation.
This genomic region on chromosome I also contains other
genes associated with pigmentation. Next to the PKS ALB1 is
a putative multicopper oxidase (MGG_ 07220.5), which is
potentially orthologous to a spore pigmentation related gene
in A. fumigatus, abr1. The signal intensity of MGG_ 07220.5
was low, but showed a significant increase during appresso-
rium initiation. This region also includes a putative threonine
deaminase (MGG_ 07224.5) and a putative peptide trans-
porter (MGG_ 07228.5), both of which exhibited up-regu-
lated expression in the appressorium consensus gene set. The
melanin biosynthesis gene cluster in A. fumigatus also con-
tains the genes for yellowish-green 1 (ayg1) and a laccase
(abr2) in addition to alb1 (PKS), arp1 (SCD) and arp2 (THR)
. MGG_ 12564.5 is a hypothetical protein with 55% iden-
tity and 70% similarity to ayg1 and MGG_ 08523.5 has 36%
identity and 55% similarity to abr2. Both MGG_ 12564.5 and
MGG_ 08523.5 were significantly up-regulated during
appressorium formation, although signal intensity was low.
However, in contrast to reduced spore pigmentation in ayg1
and abr2 deletion mutants in A. fumigatus, targeted gene dis-
ruption mutants of MGG_ 12564.5 or MGG_ 08523.5 in M.
oryzae resulted in no obvious phenotypic changes, including
appressorium pigmentation and pathogenicity on barley
plants compared to wild type (Figure 6).
In addition to the PKS ALB1 required for melanin biosynthe-
sis, several other PKS genes involved in possible toxin biosyn-
thesis were induced during appressorium formation.
Increased gene expression was found for the putative PKS
MGG_ 04775.5, which appears to be the ortholog of PKS1 and
PKS2 required for T-toxin production in the maize leaf blight
fungus Cochliobolus heterostrophus. The genomic neighbor-
hood around MGG_ 04775.5 contains a serine hydrolase
(MGG_ 04774.5), an ABC transporter (MGG_ 13762.5), and a
polyphenol oxidase (MGG_ 13764.5). These clustered genes
were all up-regulated in the appressorium consensus gene set
except MGG_ 13762.5, which was only up-regulated on the
hydrophobic surface. Similar to the Tox1 locus in C. heteros-
trophus , which contains two PKS genes, MGG_ 04775.5
was found to be closely located with another PKS1 homolog,
MGG_ 13767.5. However, this gene exhibited no significant
changes in gene expression. Expression of MGG_ 07803.5,
another PKS gene, also did not change significantly; however,
several neighboring genes, MGG_ 07784.5, MGG_ 07785.5, a
manganese peroxidase (MGG_ 07790.5), an oxidoreductase
(MGG_ 07793.5) and a major facilitator superfamily trans-
porter (MGG_ 07808.5) were induced during appressorium
formation. It is possible this cluster of genes directs the syn-
thesis of an unknown secondary metabolite.
Transcript levels of MGG_ 10072.5, which has 69% identity
with PKSN required for alternapyrone biosynthesis in the
early blight fungal pathogen Alternaria solani, was dramati-
cally increased during appressorium maturation and cAMP
treatment (52- and 100-fold change, respectively). Interest-
ingly, the genomic region containing MGG_ 10072.5 also con-
tains a putative FAD-dependent oxygenase (MGG_ 13597.5)
and two cytochrome P450s (MGG_ 10070.5, MGG_ 10071.5),
which is very similar to the genomic organization containing
the PKSN locus in A. solani .
HMG-CoA synthase (3-hydroxy-3-methylglutaryl-coenzyme
A synthase) and HMG-CoA reductase (3-hydroxy-3-methyl-
glutaryl-coenzyme A reductase) catalyze the conversion of
acetoacetyl-CoA to HMG-CoA and HMG-CoA to mevalonate,
the limiting steps for the synthesis of isoprenoids, such as
cholesterol and egosterol as well as numerous secondary
metabolites, via the mevalonate pathway. Both HMG-CoA
synthase (MGG_ 01026.5)
(MGG_ 08975.5) were up-regulated in the appressorium con-
sensus gene set. Intriguingly, a PKS (MGG_ 08969.5), a regu-
latory enzyme (MGG_ 08974.5) and a secondary metabolite
transporter (MGG_ 08970.5) flank MGG_ 08975.5. The
expression levels of these genes were not significantly
changed. However, in other fungi that contain similar
arrangements of apparently orthologous genes, these genes
confer important regulation and biological properties. In
Penicillium citrinum, the orthologous genomic region con-
tains a cluster of genes that synthesize ML-236B (compactin),
a lovestatin-like inhibitor of HMG-CoA reductase . Fur-
thermore, MGG_ 08969.5 appears orthologus to NPS6, a
gene required for fungal virulence and resistance against oxi-
dative stress in plant pathogenic ascomycetes fungi [76,77],
suggesting that this gene cluster may play an important role
in the pathogenicity of M. oryzae.
and HMG-CoA reductase
Several other genes involved in secondary metabolism were
found in the up-regulated appressorium consensus gene set.
For example, MGG_ 00385.5 and MGG_ 00573.5 encode pro-
teins homologous to an ochratoxin-A non-ribosomal peptide
synthetase in Penicillium tetracenomycin and an O-methyl-
transferase involved in polyketide synthesis in Streptomyces
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Genome Biology 2008, 9:R85
MGG_ 06585.5 and MGG_ 04911.5, which are respectively
similar to a putative short-chain dehydrogenase/reductase,
Fum13p, and a putative cytochrome P450 monooxygenase,
Fum15p, in the fumonisin biosynthesis gene cluster of the
maize ear rot fungus Gibberella moniliformis. Genes encod-
ing other key enzymes catalyzing critical steps in secondary
metabolism were also up-regulated. For example, phenyla-
lanine ammonia lyase (PAL; MGG_ 10036.5), which catalyzes
the non-oxidatative deamination of phenylalanine to cin-
namic acid and ammonia, the opening reaction in the phenyl-
propanoid pathway that generates various precursors of
secondary metabolites such as toxins, antibiotics and pig-
ments, was in the set of up-regulated appressorium
consensus genes. Other important genes for secondary
metabolism such as squalene
(MGG_ 00792.5), a key enzyme for hopanoid (triterpenoid)
biosynthesis and isoflavone reductase (MGG_ 06539.5), a key
enzyme in phenylpropanoid biosynthesis, were up-regulated.
respectively. Other examples include
The identification of several genes central to secondary
metabolism with elevated expression strongly implies the
existence of appressorium specific fungal metabolites that
may play a role in establishing the pathogenic interaction. For
example, it has been shown that the host selective toxin (HC-
toxin) synthesis is highly induced during appressorium devel-
opment in the maize leaf spot pathogen Cochliobolus carbo-
num [78,79]. To investigate the role of secondary metabolites
in appressorium formation and function, we selected two key
genes for functional analysis, the PKS ortholog of PKS1
(MGG_ 04775.5) required for T-toxin in C. heterostrophus
and the PAL gene (MGG_ 10036.5). However, unlike PKS1
and PKS2, which are required for T-toxin production and
high virulence of C. heterostrophus , targeted knock out
mutants of MGG_ 04775.5 were indistinguishable from the
wild type, were able to form appressoria and were pathogenic
towards barley and rice. Likewise, deletion mutants of
MGG_ 10036.5 appeared to have a normal phenotype in
growth, development and pathogenicity compared with the
wild type (Figure 6). PAL was also found to be dispensable for
sexual development and virulence in the maize smut fungal
pathogen Ustilago maydis .
Fungal transporters play an essential role in pathogenicity by
exporting host-specific and non-host-specific secondary
metabolites, including toxins, into the host plant tissue or
provide a protective role by removing plant defense com-
pounds or disease control agents from the fungal cell .
During appressorium development, the expression of 35
transporters with various substrate specificities were differ-
entially up-regulated with the most striking being transport-
ers related to toxin export and ion transport.
During appressorium induction, gene expression of 11 trans-
porters was increased whereas in mature appressoria, 25
transporters were up-regulated. Up-regulated transporters
included several from the major facilitator superfamily, such
as MGG_ 02167.5, MGG_ 01778.5,
MGG_ 06794.5, MGG_ 03640.5 and MGG_ 03843.5, which
share close homology with toxin efflux pumps and multidrug
transporters as well as MGG_ 07062.5 and MGG_ 09827.5,
and MGG_ 04511.5 and MGG_ 00275.5, which appear to be
involved in exporting monocarboxylate and nicotinamide
mononucleotide, respectively. Other up-regulated transport-
ers included the ABC transporter (MG10410.5), which is
homologus to mdrA2 in Dictyostelium discoideum, and
MGG_ 06604.5, a homolog of human CLN3, which is involved
in the ATP-dependent transport of arginine into the vacuole,
and putative peptide trasporters MG10200.5, MGG_ 07228.5
and MGG_ 08258.5. In contrast, gene expression of several
putative carbohydrate transporters
MGG_ 09193.5, MGG_ 03298.5,
MGG_ 08968.5) was down-regulated, as might be expected
due to the lack of nutrients in the surrounding environment.
MGG_ 07843.5, and
Enhanced gene expression was also detected in a group of
putative ion transporters, such as a K+ transporter
(MGG_ 02124.5), a cation efflux pump (MGG_ 07494.5), a
CorA-like Mg2+ transporter (MGG_ 02763.5), P-type ATPases
(MGG_ 00930.6 and MGG_ 04852.5) and other ion trans-
porters (MGG_ 05085.5 and MGG_ 04105.5). A putative large
conductance mechanosensitive channel (MGG_ 01489.5) was
down-regulated during appressorium formation.
Several fungal transporters have been recognized to play a
role in cellular development and are regarded as virulence
factors. In M. oryzae, an ATP driven efflux pump, ABC1
(MGG_ 13624.5) was strongly induced by azole fungicides
and the rice phytoalexin sakuranetin. Mutants lacking ABC1
were unable to colonize host tissue . Likewise, deletion of
the multidrug resistance transporter ABC3 (MGG_ 13762.5)
led to complete loss of pathogenicity, although appressorium
formation was unaffected . Deletion mutants of Pde1, a P-
type ATPase, were impaired in appressorium development
and pathogenicity . In our experiments, no significant
changes in gene expression of ABC1 and Pde1 were detected
MGG_ 04852.5, the closest homolog of Pde1 (50% identity)
was present in the up-regulated appressorium consensus set.
ABC3 was highly induced during early stages of appressorium
development on the hydrophobic surface.
Elevated vesicle transport and secreted proteins
The vesicular secretory pathways have not been well studied
in plant pathogenic fungi; however, increased expression of
genes involved in membrane trafficking and signal transduc-
tion was apparent during appressorium formation. Up-regu-
lated genes included a homolog of yeast phosphatidylinositol
transfer protein, Sec14p, for vesicle budding from the Golgi
complex (MGG_ 00871.5), a putative phospholiphase-D for
coated vesicle formation (MGG_ 05804.5), a putative
Genome Biology 2008, 9:R85
Genome Biology 2008, Volume 9, Issue 5, Article R85 Oh et al. R85.16
dynamin GTPase for vesicle detachment from membrane
(MGG_ 08732.5), and putative lipid binding proteins with a
C2 domain, a Ca2+-dependent membrane-targeting module
for signal transduction
(MGG_ 09947.5 and MGG_ 01150.5). Another putative trans-
membrane protein with a C2 domain, MGG_ 01094.5, was
down-regulated. The up-regulation of a number of genes
associated with vesicle transport and secretion is consistent
with our observation that expression of secreted proteins was
enriched during appressorium formation.
or membrane trafficking
Secreted proteins are likely to be key determinants of host
fungal pathogen interactions [84-88]. The overall percentage
of putative secreted proteins in the M. oryzae proteome is 7%.
However, about 26% of appressorium consensus genes con-
tain proteins with translocation signals and include several
previously characterized pathogenicity-related genes such as
GAS (gEgh16 homologs expressed in appressorium stage)
homologs and hydrophobin proteins. GAS3 (MGG_ 04202.5)
and GAS1 (MGG_ 12337.5) were previously shown by
differential hybridization analysis to be highly abundant in an
appressorium-specific cDNA library . Deletion mutants
developed appressoria but showed reduced pathogenicity on
rice and barley leaves . We found that both GAS1 and
GAS3 were highly up-regulated throughout appressorium
formation. In addition, other GAS homologs (MGG_ 02253.5
and MGG_ 09875.5) were also differentially expressed. Dur-
ing appressorium morphogenesis, MGG_ 09875.5, the closest
paralog to GAS1 (62% identity), was strongly down-regulated
(0.1-, 0.2-, 0.2-fold change in AI, AM and CI, respectively)
while MGG_ 02253.5, the closest paralog to GAS3 (45% iden-
tity) was down-regulated during AI but up-regulated in
mature and cAMP induced appressoria (0.3-, 2.5-, 2.7- in AI,
AM and CI, respectively).
The hydrophobin MPG1 (MGG_ 10315.5) was previously
shown to be highly expressed in infected leaves compared to
growth in complete media. Further, deletion mutants pro-
duced less appressoria and exhibited reduced pathogenicity
. In this study, similar to PTH11, gene expression of MPG1
was strongly down-regulated on the hydrophobic surface and
by exogenous cAMP treatment compared to germ tubes that
continued to develop vegetatively. We also observed that the
expression profile of another extracelluar protein SnodProt1
homolog (MGG_ 05344.5), which has some physical proper-
ties in common with hydrophobins, was also down-regulated
during appressorium formation. SnodProt1 is a member of
the cerato-platanin family, which in Phaeosphaeria nodorum
was shown to be involved in pathogenicity and plant defense
response in wheat . Recently, the SnodProt1 homolog in
M. oryzae has been functionally characterized . Targeted
deletion mutants exhibited reduced pathogenicity and
impaired in planta growth, but there was no evidence that
purified SnodProt1 protein had phytotoxic properties as sug-
gested for other fungal homologs .
Other secreted proteins have hydrolytic enzyme activity
potentially involved in degradation of the host cell wall and
may play some role in appressorium development and func-
tion. For example, putative
(MGG_ 02393.5, MGG_ 11966.5) and a fungal lignin peroxi-
dase (MGG_ 07790.5) were highly up-regulated. Until
recently the role of cutinase was largely discounted based on
the finding that the cutinase CUT1 (MGG_ 01943.5) is not
essential for pathogenicity in M. oryzae . However,
expression of this gene was not up-regulated in our experi-
ments. Skamnioti and Gurr  reported that CUT2
(MGG_ 09100.5) is required for full virulence. As reported by
Skamnioti and Gurr , we also found this gene was highly
up-regulated during late stages of appressorium formation on
the hydrophobic surface. To further evaluate the role of cuti-
nases, we investigated the function of the putative cutinase
MGG_ 02393.5 because this gene was also up-regulated by
both hydrophobic and cAMP. However, gene deletion
mutants exhibited no observable changes in virulence or
other phenotypic differences (Figure 6).
Cell signaling pathways
In addition to cAMP, several cell signaling pathways have
been shown to be involved in regulating appressorium forma-
tion. Calcineurin is a Ca2+/calmodulin-dependent serine/
threonine phosphatase that is involved in many signal path-
ways for cation homeostasis, cell differentiation, morphogen-
esis, cell wall integrity and pathogenicity [95-98].
Phosphorylation activity of calcineurin is inactivated when it
is coupled with cyclophilin and other FK506-binding proteins
(FKBPs) in the presence of inhibitors such as cyclosporine A
and tacrolimus (FK506). In M. oryzae, cyclophilin (CYP1;
MGG_ 10447.5) has been shown to be involved in fungal viru-
lence and appressorium turgor generation . Cyclosporine
A inhibits hyphal growth and appressorium formation in a
CYP1-dependent manner, supporting a role for calcineurin in
regulating appressorium development. The transcriptional
activity of calcineurin (MGG_ 07456.5) remained unchanged;
however, expression of MGG_ 06035.5, a putative ortholog of
FKBP12, decreased. In addition, MGG_ 01150.5, a putative
ortholog of CTS1 (calcineurin temperature suppressor),
which is required for the full virulence of Crytococcus
neoformans, was up-regulated
Many external signals are transmitted to the inner workings
of the cell through receptors that are embedded in the plasma
membrane. During appressorium induction, several genes
encoding putative G-protein coupled receptor-like proteins
with seven membrane spanning
MGG_ 05871.5, MGG_ 02692.5,
MGG_ 10571.5) were significantly down-regulated, although
the expression of a
(MGG_ 09570.5; CFEM is an eight cysteine-containing
domain present in fungal extracellular membrane proteins)
was up-regulated. Also, expression of another CFEM protein,
Genome Biology 2008, Volume 9, Issue 5, Article R85 Oh et al. R85.17
Genome Biology 2008, 9:R85
ACII (MGG_ 05531.5), which interacts with MAC1 adenylate
cyclase in cAMP signaling pathways, was very intensive and
was up-regulated in young appressoria but was dramatically
reduced in mature appressoria and was significantly down-
regulated by cAMP. We also observed increased gene expres-
sion of MGG_ 00438.5, which encodes a putative transmem-
brane protein, PAT 531. Previous studies reported that
deletion of this gene resulted in reduced pathogenicity of M.
oryzae towards weeping lovegrass .
In this study, we performed microarray studies to identify
genome wide fluctuations in gene expression during
germination, appressorium induction and maturation. We
then used these data as a guide to identify and subsequently
characterize core biological processes, in some cases previ-
ously unrecognized processes required for infection related
development and pathogenesis. Key to this study was the
experimental design. First, we performed a direct comparison
of gene expression during spore germination on an inductive
surface verses a non-inductive surface. Second, we compared
the expression patterns with those obtained from a direct
comparison of conidia germinating in the presence of cAMP
verses its absence. The integration of these data sets revealed
a core set of appressorium-induced genes common to the dif-
ferent stimuli. Subsequent bioinformatics and functional
analyses of these so-called consensus genes shed new light on
the biochemical processes required for appressorium forma-
tion and function.
Prior to this study, no genome-wide transcriptional profiles
related to infection-related morphogenesis have been pub-
lished for filamentous fungi. A few studies using partial gene
sets have provided some insights into fungal pathogenesis
[41,42,101,102]. However, due to a number of reasons,
including experimental design, scale of experiments, analytic
procedures applied, as well as lack of supporting functional
evaluation, drawing generalized conclusions must be
approached cautiously. Nevertheless, our data reveal a
number of key features and represents an important bench-
mark for other future studies of pre-infection-related devel-
opment. Very little had been known regarding the role of
protein degradation and amino acid metabolism in appresso-
rium formation and function. We found that during appresso-
rium formation a number of genes required for protein
metabolism were differentially expressed at significant levels.
In particular, several proteases, including the putative vacu-
olar subtilisin-like protease SPM1, which is required for pen-
etration and in planta growth, were up-regulated specifically
during appressorium formation. Our data also revealed that
genes involved in targeting proteins for degradation as well as
the machinery involved in protein degradation were also up-
regulated. Several proteases contained an export signal, sug-
gesting they may be secreted and act as virulence factors as
has been shown for homologs in related fungi . Coupled
with elevated expression of genes for protein degradation, we
found genes involved in amino acid metabolism to be stimu-
lated during appressorium formation. For example, a NAD-
dependent glutamate dehydrogenase (Mgd1) was signifi-
cantly up-regulated in a manner very similar to SPM1. Mgd1
deletion mutants were unable to make normal appressoria
and showed significantly reduced virulence. Microarray
analysis of 1,730 genes from the insect fungal pathogen
Metarhizium anisopliae also revealed up-regulation of a
number genes involved in protein metabolism, including sev-
eral proteases, as well as amino acid catabolism during
appressorium formation on insect cuticle [102,104,105]. It
should be pointed out that proteins represent a major compo-
nent of insect cuticle and, thus, induction of proteases was not
unexpected. Several proteases, including subtilisins, were
also up-regulated during starvation conditions, but less so
than on insect cuticle . Others were specifically up-regu-
lated on cuticle. The authors speculate that proteases may
'sample' the cuticle, resulting in further induction for nutri-
tion and possibly to aid in entering the host. This, however,
has not been formally demonstrated. Also, similar to our
observations in M. oryzae, an NAD-GDH was up-regulated in
M. anisopliae on insect cuticle, possibly in response to ele-
vated levels of amino acids released through protein hydroly-
sis and uptake. In yeast, NAD-GDH is repressed by
ammonium and derepressed by glutamate and carbon starva-
tion [49,106,107]. However, iHOncreased enzyme activity
was observed during yeast to mycelium morphogenesis in
Mucor racemousus and Benjamineilla poitrasii and hyphal
adhesion and arial hyphae development in Neurospora
crassa [108-110], suggesting NAD-GDH plays a critical role
in development. In sum, for M. oryzae, the combination of
global gene expression analysis and subsequent functional
interrogation revealed a direct connection between protein
catabolism, carbon and nitrogen scavenging from amino
acids and appressorium formation and function.
In recent years, attention has focused on effector molecules,
many of which are secreted proteins or secondary metabo-
lites. These molecules may serve as virulence determinants or
help shield the fungus from being detected by its host. In
some cases, the host may recognize these molecules and acti-
vate its defense mechanisms. For example, the M. oryzae
avirulence gene ACE1 encodes a PKS [85,111]. In this case, it
is presumed that the metabolite synthesized by this gene
product confers biological function. Other avirulence genes
such as avr-pita encode a small-secreted protein that has
been shown to directly interact with its cognate resistance
gene product, Pita, in rice . Secreted effector proteins
identified in oomycetes share the common motif, RXLR,
which is essential for their translocation into host plant cells
. Enriched in the apoplast of tomato leaves, the chitin
binding protein Avr4 from Cladosporium fulvum protects the
fungal cell wall from the attack by plant chitinases . Dur-
ing colonization of tomato xylem vessels, the root invading
fungus Fusarium oxysporum secretes a number of small
Genome Biology 2008, 9:R85
Genome Biology 2008, Volume 9, Issue 5, Article R85 Oh et al. R85.18
cysteine-rich proteins, such as SIX1, which are recognized by
host resistance gene products . Whole genome expres-
sion studies using the cereal head blight fungus Fusarium
graminearum Affymetrix GeneChip revealed numerous
genes encoding potential plant cell wall degrading enzymes,
including xylanases, mannanases, pectinases, glucanases,
galactosidases and cutinases, as well as genes involved in tri-
chothecene biosynthesis, were expressed in planta .
Thus, it is reasonable to expect that during pre-penetration
phases of the host-pathogen interaction, M. oryzae would
begin to mobilize effector molecules. Indeed, during appres-
sorium formation, we detected a nearly four-fold enrichment
of genes encoding products with a putative signal peptide in
the up-regulated consensus gene set as well as increased
expression of numerous genes associated with secondary
metabolism and secretion. GAS1 and GAS3 were among the
most highly expressed secreted proteins. Stage-dependent
gene expression of GAS homologs was previously reported in
the cereal powdery mildew fungus Blumeria graminis.
Egh16H1 was strongly induced during haustorium formation
whereas gEgh16 appears to be more induced during spore
germination . The function of these genes in B. graminis,
however, has not been determined. A number of secreted pro-
teins, predominantly hydrolases such as proteases and chiti-
nases, were up-regulated during appressorium formation by
M. anisopliae on insect cuticle . Although these enzymes
are likely involved in nutrition, it is possible they release
effector molecules. Chitinase and parts of the secretion appa-
ratus were also found to be up-regulated in B. graminis dur-
ing the pre-penetration stages compared to conidial
germination, which presumably would promote delivery of
any effector molecules . Indeed, a number of genes
encoding small secreted metallothioneins were found to be
strongly expressed during haustoria formation by the rust
fungus Uromyces fabae compared to germinated ure-
dospores . We also found several examples of co-
expressed genes clustered around PKS genes, suggesting they
may synthesize pathogenesis-associated metabolites. Several
phytotoxic secondary metabolites have been isolated from M.
oryzae, such as pyricularin and picolinic acid, but little is
known regarding the genes that are required for their synthe-
sis . This is a research topic that would appear to warrant
Somewhat surprisingly, some genes known to be essential for
appressorium development appeared to be down-regulated
in our expression studies. The importance of MPG1
(MGG_ 010315.5), a hydrophobin,
(MGG_ 05871.5), a membrane spanning protein in appresso-
rium development, are well documented, but our data
showed they were more highly expressed in spores germinat-
ing on a hydrophilic surface than in incipient appressoria.
This may suggest that the gene products are required initially
and perhaps transiently for subsequent morphological
changes and is consistent with a role in surface sensing and
attachment. Once the environmental cue is detected and the
intracellular signal pathways activated, these proteins may no
longer be needed for appressorium formation to proceed.
In addition, our data also revealed that a large set of ribos-
omal protein genes were down-regulated during appresso-
rium formation. Similar observations were reported for M.
anisopliae during appressorium formation on insect cuticle
and in the animal pathogens C. neoformans and Candida
albicans early in pathogenesis following internalization by
macrophages [104,117,118]. This pattern of down-regulation
of genes associated with ribosome biogenesis has been
observed commonly in a number of organisms subjected to
nutrient starvation or upon treatment with rapamycin, a
potent inhibitor of target of rapamycin (TOR) kinase . In
M. oryzae, this set of genes, with the exception of
MGG_ 05716.5, was down-regulated significantly (>2-fold)
when cells were shifted to media lacking nitrogen for 12 hours
. In the experiments reported here, spores were germi-
nated in water and thus would be starved for both nitrogen
and carbon; however, it was only upon appressorium forma-
tion that expression of genes associated with ribosome bio-
genesis fell. The role of TOR is complex, it is not only
associated with starvation but with development. In yeast,
TOR interacts with a number of other proteins, including
STE20, and regulates sexual development . In addition,
rapamycin is a potent inhibitor of filamentous growth in
fungi, including A. fumigatus . Thus, during appresso-
rium formation, the TOR pathway may be involved in redi-
recting resources away from polar growth to breaking down
cellular components in order to generate materials necessary
for the penetration process to be effective.
While perturbation of any one signal cascade is often suffi-
cient to affect appressorium formation, considerable evi-
dence suggests that there is cross-talk between the signal
pathways. Previous studies have shown that expression of
GAS1 and GAS2, which are highly expressed during appresso-
rium formation, requires Pmk1, suggesting they are regulated
by the MAPK pathway . The presence of these genes in
the appressorium consensus gene set is consistent with cross-
talk between the cAMP and MAPK pathways. In addition, our
microarray data suggest the involvement of the calcineurin
dependent and TOR signal pathways in regulating appresso-
rium formation. Clarifying the role and interconnection of
these different signal pathways may be served by examining
gene expression in different mutant backgrounds, particu-
larly within the first few hours of germination.
While gene expression analysis is a useful tool to identify
genes associated with a particular process, it is by no means
definitive. In a recent report, 250 M. oryzae mutants defec-
tive in appressorium formation and/or pathogenesis were
created by agrobacterium-mediated random insertion muta-
genesis . The majority of the mutation sites were located
in intergenic regions. Interestingly, most of the genes flank-
ing the insertion sites showed no differential expression
Genome Biology 2008, Volume 9, Issue 5, Article R85 Oh et al. R85.19
Genome Biology 2008, 9:R85
changes in our microarray experiments. Moreover, of 90
genes with well-characterized roles in pathogenicity in M.
oryzae, the vast majority (81) were not up-regulated during
appressorium formation. For example, lipid metabolism has
been strongly linked to generating the high levels of glycerol
found in appressoria and triacylglycerol lipase activity has
been reported to be induced during appressorium formation
. However, in our microarray study, only one
(MGG_ 00528.5) of seven genes encoding this activity
showed any evidence of induction and even then expression
levels were low. In addition, we observed that MFP1, the gene
that encodes a multifunctional beta-oxidation protein
involved in lipid degradation, was more highly expressed in
spores than during germination or appressorium formation.
Also, homologs of genes involved in peroxisome biosynthesis,
such as MgPex6, exhibited little indication of being up-regu-
lated during appressorium formation. Similarly, we found
limited evidence for genes involved in autophagy being up-
regulated during appressorium formation. Both MgATG1 or
MgATG8 were induced slightly during germination, but
showed little or no further induction during appressorium
On the other hand, the majority of mutants generated in our
study, which were genes highly up-regulated during appres-
sorium formation, retained their ability to differentiate infec-
tion structures and cause disease. It is possible that the lack of
an observable phenotype variant in many of our gene knock-
out mutants may be due to functional redundancy among
possible (unidentified) paralogs or the activation of alterna-
tive compensatory pathways. For example, M. oryzae
appears capable of compensating for the loss of
MGG_ 02393.5, but appears to be unable to fully compensate
for the loss of CUT2, even though both homologs exhibited
similar expression patterns. Deleting SPM1 had a dramatic
effect on sporulation and virulence, but deleting its paralog,
MGG_ 09246.5, showed no detectable phenotypic changes
despite both genes having very similar expression patterns.
Thus, M. oryzae is able to fully compensate for the loss of
some gene homologs but not others. This suggests some level
of functional specificity may exist. Functional redundancy/
compensation appears to be commonly encountered in bio-
logical processes. For example, only about 10% of genes
down-regulated by genome-wide RNA interference provided
phenotypic changes in Caenorhabditis elegans . In S.
cerevisiae, less than 7% of genes that exhibited a significant
increase in gene expression were found to be required for
optimal growth under the tested conditions. Moreover, many
genes that were necessary for the fitness were not differen-
tially expressed. . To address issues related to functional
redundancy in M. oryzae, employing RNA interference to
silence all members of a gene family may be effective. In other
situations, it may be valuable to explore the use of over-
expression to evaluate the role of particular genes.
Genome-wide expression profiling during spore germination
and appressorium formation revealed that the blast pathogen
M. oryzae undergoes significant changes in gene expression
during infection-related cellular differentiation. Functional
analysis and characterization of the differentially expressed
genes provides new insight
morphogenesis, in particular regarding the role of protein
degradation for appressorium function. We provide a com-
prehensive list of genes that might be involved in appresso-
rium formation and function solely or in combination with
other genes. Our data will be beneficial for further studies on
fungal pathogenesis, including gene expression studies in
other fungal pathogens. We expect the emergence of addi-
tional functional information regarding M. oryzae genes with
currently no known biological function will help broaden the
scope of future analyses.
Materials and methods
Appressorium induction by physical cue and exogenous
M. oryzae strain 70-15 spores were collected from ten-day-
old V8 agar plates and adjusted to 105 conidia/ml. Spore sus-
pensions were placed on the appressorium-inducing (hydro-
phobic) and non-inducing (hydrophilic) surface of GelBond
(Cambrex Bio Science Inc., Rockland, ME, USA) at 6.25 × 104
spores/cm2 and incubated at 25°C in the dark and for 7 and 12
h. By 7 h, on the hydrophobic side of the GelBond film, essen-
tially all spores germinated and tips of germ tubes had started
to hook and swell, forming young appressoria. These young
light-colored appressoria developed into dark mature appres-
soria at 12 h. On the hydrophilic side of the GelBond, germ
tubes continuously elongated and branched several times
through 12 h with no other developmental changes. Appres-
soria were also induced by adding exogenous cAMP (final
concentration of 50 mM) to the spore suspension placed on
the hydrophilic surface of GelBond as described above. In the
presence of cAMP, melanized appressoria were evident at 9 h
RNA sample preparation and microarray data
RNA was purified following standard protocols. Briefly, fol-
lowing incubation, fungal material was flash frozen with liq-
uid nitrogen, scrapped from the support, and ground in liquid
nitrogen. Total RNA was extracted using Qiagen RNAeasy
Mini kit (Qiagen Inc., Valencia, CA, USA) according to the
manufacturer's protocol. RNA was also extracted from spores
in water immediately after being collected from agar plates.
Quality analysis and quantification were performed using the
Agilent Bioanalyzer (Agilent Technologies, Inc., Wilmington,
DE, USA) and the Nano Drop (NanoDrop Technologies, Inc.,
Wilmington, DE, USA).
Genome Biology 2008, 9:R85
Genome Biology 2008, Volume 9, Issue 5, Article R85 Oh et al. R85.20
RNA from the two biological replicates was pooled in equal
amounts and 1 μg was labeled by reverse transcriptional
incorporation of cyanine 3 (Cy3) or cyanine 5 (Cy5) labeled
dCTP, using an oligo(dT) primer. The Agilent Fluorescent
Linear Amplification Kit protocol for fluorescent cRNA syn-
thesis was used. The concentration and the dye incorporation
of labeled cRNA were measured by Nano Drop. According to
the microarray hybridization scheme (Figure 1b), equimolar
amounts of cRNA samples, each labeled with Cy3 or Cy5,
were co-hybridized to the M. oryzae Oligo Microarray (Agi-
lent technology, #G4137A) using the Agilent 60-mer-oligo
microarray processing protocol as previously described .
Following drying, slides were immediately scanned with an
Agilent G256BA microarray scanner. Image files from the
scanner were analyzed with the Agilent 2567AA Feature
Extraction software (version 18.104.22.168). For signal normaliza-
tion, the output from Agilent Feature Extraction was first
converted into GPR format to conform to the input format
requirements for the 'Bioconductor/Aroma' software .
The data were read into Aroma and 'within slides LOWESS
normalization' and 'across slides LOWESS normalization'
methods were applied. After normalization, the data were
extracted for each treatment - each having four replicates, two
from Cy3 and two from Cy5, from which the mean and other
statistics were calculated.
Gene expression profiles and correlation analysis
Gene expression profiles for appressorium initiation, appres-
sorium maturation and appressorium induced by cAMP were
determined by comparing the hybridization signals of RNA
harvested from the hydrophobic surface after 7 and 12 h incu-
bation (Pho7 and Pho12, respectively) and the hydrophilic
surface after 9 h incubation with cAMP (cAMP9), directly to
those from the hydrophilic surface after 7 and 12 h incubation
(Phil7 and Phil12, respectively), and the hydrophilic surface
after 9 h incubation without cAMP (Phil9). The expression
profile during germ tube elongation was made by comparing
the hybridization signals of RNA harvested from the
hydrophilic surface after 12 h (Phil12) to those from the
hydrophilic surface after 7 h (Pho7). To construct the gene
expression profile for spore germination, the hybridization
signals of RNA harvested from the hydrophilic surface after 7
h incubation (Phil7) were compared to those of RNA from
ungerminated spores (Spore). For each profile, the ratio of
the expression level for the 10,176 M. oryzae probes was cal-
culated. The log2 -transformed value of the ratio was used for
pairwise correlation analysis between expression profiles.
Genes were designated as differentially expressed if their
average signal intensities were equal or above 200 in at least
one condition and their expression ratios were equal or
greater than 2-fold with p < 0.05 (Student's t-test).
RT-PCR and quantitative RT-PCR
RT-PCR as described previously was conducted on selected
genes that were either significantly up-regulated, down-regu-
lated, or showed no difference in their expression profile in
the microarray experiments . PCR was performed on
independently generated templates using primers shown in
Additional data file 2. Total RNA was prepared from germi-
nating spores on hydrophilic and hydrophobic GelBond sur-
faces using the RNAeasy Plant Mini Kit (Qiagen Inc.) and
used for reverse transcription. cDNA was prepared according
to procedures for cDNA synthesis from total RNA in the Low
RNA Input Fluorescent Linear Amplification Kit (Agilent
Technologies, Inc.) with modification.
For qRT-PCR, total RNA was independently extracted from
cAMP induced appressoria on the hydrophilic surface of Gel-
Bond as well as germinating spores without cAMP treatment,
and cDNA was synthesized as described above. qRT-PCR
reactions with MPG1 and PTH11 were carried out using 2×
SYBR Green Master Mix (Applied Biosystems, Foster City,
CA, USA) with that of the actin gene as an internal control.
qRT-PCR was performed using the following profile: 94°C for
3 minutes; 30 cycles at 94°C, 45 s; 62°C, 45 s; 68°C, 2 min-
utes; and a final extension at 68°C for 7 minutes. Analysis of
the qRT-PCR results was carried out as previously described
by Livak and Schmittgen .
Gene Ontology and functional annotation
Orthologs were identified between M. oryzae predicted pro-
teins and proteins in the GO database  via searching
reciprocal best hits with the following cut-offs; e-value, 1.0e-
3, and identity, 20%. Results from local alignment using
BLAST, functional domain comparisons from Interpro and
prediction of signal peptides from SignalP 3.0 software and a
manual literature review were used to make final assignments
to GO functional categories.
Targeted gene replacement and mutant screening
Gene replacement cassettes were constructed using
adaptamer mediated PCR (Additional data file 5) . Typ-
ically, 1.3 kb of upstream and downstream sequence of each
target gene was amplified with primers that contained
adaptamer sequences (Additional data file 6). A 1.5 kb frag-
ment containing the hygromycin dehydrogenase gene with
the trpC promoter from A. nidulans was amplified from plas-
mid PCB1003 using the adaptamer sequence attached to the
forward HPHF and reverse HPHR primer set. Using nested
primers (Additional data file 6) from inside of the 5' upstream
fragment and from inside of the 3' end of the downstream
fragment of the target gene, the individual fragments and
hygromycin resistance gene fragment were combined and
amplified together to construct a hygromycin cassette for
gene replacement typically approximately 3.3 kb in length.
The hygromycin cassette was transformed into 70-15 proto-
plasts as previously described . Gene replacement
mutants were identified by PCR screening (Additional data
file 7) and further confirmed by Southern blot analysis.
Genome Biology 2008, Volume 9, Issue 5, Article R85 Oh et al. R85.21
Genome Biology 2008, 9:R85
Mutant phenotype assays
A series of phenotype analyses were conducted on several
knockout mutants (≥ 3) and ectopic (≥ 2) transformants for
each gene functionally characterized. Germination and
appressorium assays were conducted using spores collected
from ten-day-old V8 agar plates and adjusted to 105 spores/
ml. Spore suspension was spotted on the hydrophobic and
hydrophilic surface of GelBond film and the rate of germina-
tion and appressorium formation was measured after 24 h
incubation at 25°C in the dark. To test for pathogenicity, bar-
ley and rice seedlings were spray inoculated with M. oryzae
spore suspension (3 × 104 spores/ml, Tween 20 0.025%) and
incubated in dark humid conditions at 25°C. The number and
size of lesions were recorded seven days post-inoculation.
Growth rate assays were conducted by placing 10 μl of spore
suspension (3 × 104 conidia/ml) on agar plates with complete
or minimal media. In other growth rate assays, where
minimal media was amended with glutamate and glutamine
as a sole nitrogen source, glucose was also reduced to 0.125%.
Colony morphology and diameters were recorded periodi-
cally for 15 days. The total number of spores on minimal
media plates was counted after 15 days incubation. All
experiments were conducted in triplicate and performed at
least three times.
ABC, ATP-binding cassette; AI, appressorium initiation; AM,
appressorium maturation; CI, cAMP-induced appressoria;
cAMP, cyclic AMP; FKBP, FK506-binding protein; GO, Gene
Ontology; MAPK, mitogen-activated protein kinase; NAD-
GDH, NAD-specific glutamate dehydrogenase; PAL, phenyla-
lanine ammonia-lyase; PKS, polyketide synthase; RT-PCR,
reverse transcriptase PCR; SAGE, serial analysis of gene
expression; SCD, synthalone dehydratase; THR, hydroxy-
naphthalene reductase; TOR, target of rapamycin.
YYO, TM and RAD designed the study; YYO, ND and SC car-
ried out the experiments; YYO and HP performed data
processing and statistical analysis; DB and SM created the GO
database; YYO, TM and RAD interpreted the data and wrote
Additional data files
The following additional data are available with the online
version of this paper. Additional data file 1 is a table listing GO
categorization (biological process) of differentially expressed
genes during spore germination. Additional data file 2 is a
table listing primer sequences for RT-PCR and qRT-PCR.
Additional data file 3 is a table listing categorization of
appressorium consensus genes with known function.
Additional data file 4 is a figure showing differential expres-
sion of the putative melanin biosynthesis gene cluster during
appressorium formation. Additional data file 5 is a figure
showing adaptamer mediated PCR strategy for targeted gene
deletion. Additional data file 6 is a table listing primer
sequences for gene specific replacement cassette construc-
tion. Additional data file 7 is a figure showing confirmation of
target gene replacement by PCR.
Additional data file 1GO categorization (biological process) of differentially expressed genes during spore germinationGO categorization (biological process) of differentially expressed genes during spore germination.Click here for file Additional data file 2 Primer sequences for RT-PCR and qRT-PCRPrimer sequences for RT-PCR and qRT-PCR.Click here for fileAdditional data file 3 Categorization of appressorium consensus genes with known function Categorization of appressorium consensus genes with known function.Click here for file Additional data file 4Differential expression of the putative melanin biosynthesis gene cluster during appressorium formationDifferential expression of the putative melanin biosynthesis gene cluster during appressorium formation.Click here for file Additional data file 5 Adaptamer mediated PCR strategy for targeted gene deletionAdaptamer mediated PCR strategy for targeted gene deletion. Click here for file Additional data file 6 Primer sequences for gene specific replacement cassette construction Primer sequences for gene specific replacement cassette construction. Click here for file Additional data file 7 Confirmation of target gene replacement by PCR Confirmation of target gene replacement by PCR.Click here for file
We thank Julie Macialek, Tesia Stephenson and other members of the Dean
Lab for technical assistance and Dr Sonia Herrero for help with qRT-PCR
analysis. This research was funded by a grant from the NSF Plant Genome
Research Program (#0115642).
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