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SHORT COMMUNICATION
The exceptionality of stress response in Magnaporthe oryzae: a set
of ‘‘salt stress-induced’’ genes unique to the rice blast fungus
Stefan Jacob1 • Alexander Yemelin1 • Stefan Bohnert1 • Karsten Andresen2 •
Eckhard Thines1,2
Received: 10 November 2016 / Accepted: 12 January 2017
 The Author(s) 2017. This article is published with open access at Springerlink.com
Abstract The ability of pathogens to signal perception and
adaptation to environmental changes is an important pre-
requisite for successful colonization of the host organism.
Filamentous phytopathogenic fungi, for example, have to
cope with rapid changes in the environment during inva-
sive growth in planta. Consequently, they have evolved a
range of specific factors contributing to environmental
adaptation facilitating host invasion. In addition to con-
served pathways, including genes participating in stress
response, unique/individual genes within the pathogens
might represent determinants of pathogenicity. Therefore,
identification of unique genes could provide a set of
excellent candidates for novel and specific fungicide tar-
gets. One of the environmental changes during host inva-
sion comprises the accumulation of osmolytes, which are
present in varying concentrations inside the plant. Tran-
scriptional profiling of the rice blast fungus Magnaporthe
oryzae undergoing osmotic stress revealed interesting
results. We identified a set of 239 genes which were reg-
ulated significantly by salt stress. Among these ‘‘salt stress-
regulated’’ genes, 176 (75%) of the upregulated and all of
the downregulated genes were found to have no homo-
logues in yeast when interrogation against the yeast protein
database was performed. Functional annotation analysis by
InterProScan and clustering of genes based on gene
ontology (GO) enrichment analysis was conducted to
annotate each of the ‘‘salt stress-regulated’’ genes and to
identify functional categories of biological processes
associated with environmental stress response. Finally, we
present a set of ‘‘salt stress-regulated’’ genes suggested as
unique in the rice blast fungus.
Keywords NGS  Pathogenicity  Environmental stress 
Fungicide target  Osmoregulation  Magnaporthe oryzae 
Salt stress-induced genes
Short communication
The transcriptome reflects differential gene expression
patterns by defining a dynamic link between an organism’s
genome and its physiological properties. We conducted a
comprehensive genome-wide differential expression anal-
ysis (RNA-seq) regarding 0.5 M KCl stress in the fila-
mentous phytopathogenic fungus Magnaporthe oryzae in
order to find unique gene expression profiles under ‘‘host-
mimicking’’ environmental stresses. Using salt stress as a
host-mimicking agent appears to be feasible, because salt
ion concentration of nearly 0.5 M for K? has already been
documented in plant tissues [5, 6]. The resulting ‘‘salt
stress-induced’’ genes provide a promising reservoir for
novel fungicide targets, since osmoregulation of phy-
topathogenic fungi is already known to be a suitable target
location for crop protection. The high osmolarity glycerol
pathway is a druggable signalling pathway comprising
Stefan Jacob and Alexander Yemelin have contributed equally to this
work.
Electronic supplementary material The online version of this
article (doi:10.1007/s41348-017-0074-z) contains supplementary
material, which is available to authorized users.
& Stefan Jacob
jacob@ibwf.de
1 Institut fu¨r Biotechnologie und Wirkstoff-Forschung gGmbH
(IBWF), Erwin-Schro¨dinger-Str. 56, 67663 Kaiserslautern,
Germany
2 Institut fu¨r Mikrobiologie und Weinforschung, Johannes
Gutenberg-University Mainz, Johann-Joachim-Becherweg
15, 55128 Mainz, Germany
123
J Plant Dis Prot
DOI 10.1007/s41348-017-0074-z
different target proteins [11, 13]. Thus, the identification of
genes contributing to osmoregulation or genes responsible
for adaption to environmental stresses is of high interest for
fungicide research.
We identified a total of 236 genes which were signifi-
cantly upregulated and three genes downregulated in
response to salt stress. Analysing the deduced protein
sequence homology of these ‘‘salt stress-regulated’’ genes
by interrogation of the Saccharomyces cerevisiae genome
database (www.yeastgenome.org) resulted in a set of 176
(75%) upregulated genes and all of the downregulated
genes exhibited no assignable homology to proteins in S.
cerevisiae. We focused on the representation of the 236
upregulated genes (Fig. 1a), but raw data and annotations
for the three downregulated genes are provided in the
supplementary information (Tab. S1). We checked whether
some of the upregulated candidate genes for which the
homologues in yeast were found are already known to
possess functions in stress response, osmoregulation or
even cellular signalling in S. cerevisiae to validate the data
obtained from RNA-seq analysis. Hence, we identified
several instances of well-fitted correlation with datasets
published previously. We found that the genes
MGG_01822 (MoHOG1) and MGG_10268 (MoPBS2)
were significantly upregulated when subjected to salt
stress, which is in accordance with functions published
already regarding osmotic stress regulation either in yeast
[9] or M. oryzae [4, 10]. Further evidence is given by genes
predicted to encode homologues to the yeast ATPase ENA2
(YDR039C), which is involved in salt tolerance [8, 16] or
even a group of genes involved in stress-induced transport
mechanisms (Tab. S1). The protein sequences of the ‘‘salt
stress-induced’’ genes deduced were interrogated against
the non-redundant database from the National Centre for
Biotechnology Information and Pathogen Host Interactions
(PHI) databases with the aim of identifying homologues in
other organisms and putative pathogenicity-associated
factors. The interrogation of the genes selected in the PHI
database partly confirmed the hypothesis that the salt
stress-affected genes identified may be related to
pathogenicity-related processes in filamentous fungi. The
PHI-based homology search resulted in 35 (of 236) listed
homologues in the public database, and 22 (63%) of them
have been published to be virulence or pathogenicity fac-
tors (Tab. S1). Furthermore, we addressed our hypothesis
of a putative relationship between environmental stress and
virulence by comparing the ‘‘salt stress-induced’’ genes
with Magnaporthe differentially expressed genes during in
planta growth/colonization as well as upon heat or drought
stress condition (as published by [2, 12, 14]. As a result, we
found an overlapping set of 43 genes regulated in at least
two of these conditions, but none of the genes exhibited
significant regulation in all of these conditions (provided in
Tab. S1, ‘‘conditionally regulated genes’’). Consequently,
these results indicate both functions of these genes in
similar physiological networks and involvement in differ-
ent signalling pathways. Addressing the physiological
function of the unknown ‘‘salt stress-regulated’’ genes, we
classified the candidates into different functional categories
based on Blast2GO processing of the data, including
BLASTp, InterPro and GO analysis. Thus, most of the gene
products predicted could be assigned to such functional
categories as metabolism (26), membrane/cell wall
remodelling (41) and transport (30). A functional annota-
tion for 72 of the differently expressed (DE) genes was,
however, not predictable (Fig. 1b). The GO analysis was
completed with an enrichment analysis to extract the
maximal meaning of the RNA-seq data. Further analysis
using TopGO/REVIGO refined these findings, because the
REVIGO treemap displays GO terms matching biological
processes which are appropriate for cellular response to salt
stress (Fig. 1c). As a conclusion, all these data enabled us
to present an exceptional list of 20 Magnaporthe oryzae-
unique ‘‘salt stress-upregulated’’ genes having no homo-
logues in other fungal genomes listed in public databases
(Fig. 1d). The set of specific genes will initialize future
projects studying the function of osmoregulation and
appear to be promising candidates for finding novel
fungicide targets for plant protection.
Methods
Conidia of M. oryzae were collected from eleven-day-old
complete medium agar plates [11] and adjusted to 105
conidia/ml. Spore suspensions were used as inoculum for
liquid complete medium and incubated for 48 h at 28 C at
100 rpm. These cultures were used to inoculate the main
axenic cultures, which were cultivated for a further 24 h at
28 C at 100 rpm. Total RNA from three biological
replicates was extracted as an untreated control using the
RNeasy Mini Kit (QIAGEN, plant protocol). Subsequently,
RNA was extracted from the same cultures after 25-min
treatment with 0.5 M KCl. Prior to transcriptome
sequencing, equal amounts of total RNA derived from
three biological replicates for each condition (untreated and
25 min after KCl induction) were pooled together. Library
preparation was carried out using 1.5 lg of total RNA for
each. The RNA-Seq readings were obtained from a
2 9 100 bp paired-end Illumina HiSeq 2000 single lane
run for each condition investigated, followed by a quality
check using Qualimap version 2.2 [7] and removal of
adaptor sequences. The readings retained were merged
using FLASH 1.2.9 (FLASH source: http://sourceforge.net/
projects flashpage/?source = dlp) and aligned to the ref-
erence genome of Magnaporthe oryzae 70-15 version 8
J Plant Dis Prot
123
obtained from the Broad Institute by employing TopHat
2.0.11 [18]. Expression levels for each gene were deter-
mined by using the CuffDiff quantification method [18],
and reading counts were expressed as FPKM (‘‘Fragments
Per Kilobase of transcript per Million fragments mapped’’)
values. Only those genes, having at least a fivefold change
and displaying the FPKM value of at least ten in any of the
conditions investigated, affirmed by a p value of B 0.05,
were considered as significantly differentially expressed
and were selected for further analysis. The BLASTp search
against the National Centre for Biotechnology Information
non-redundant database was conducted to identify func-
tional annotation orthologues in other fungal organisms,
using the taxonomy filter ‘‘fungi’’ (taxa 4751, Fungi) and
an E-value threshold of 1.0E-3. Furthermore, a local
BLASTp interrogation (using 1.0E-30 as an E-value cut-
off) against the Saccharomyces cerevisiae (S288C) protein
sequences obtained from the Saccharomyces cerevisiae
genome database was performed to examine the proportion
of corresponding DE genes. Additionally, gene ontology
and functional annotation of the protein sequences deduced
were employed using Blast2GO 4.0.2 [3] via searching for
Fig. 1 Illustration of the analysis of the 236 ‘‘salt stress-upregulated’’
genes in Magnaporthe oryzae. Blast2GO analysis (InterPro, BLASTp
and GO analysis) was conducted with the 236 candidate gene
sequences to refine/update the existing annotation. a Bar chart repre-
senting potential homologues in yeast, obtained from sequence
interrogation in yeast databases of significantly expressed genes
regarding salt stress (25 min, 0.5 M KCl). b Customized functional
gene categories of upregulated genes. Most of the differentially
expressed genes were found to be involved in metabolism, mem-
brane/cell remodelling or transport-associated processes. c REVIGO
treemap summarizing GO biological process categories overrepre-
sented regarding salt stress. TopGO was used to identify GO
biological processes that were overrepresented among transcripts
with increased abundance upon KCl-response at p-values of B 0.05.
Similar colours indicate semantic similarity related to similar
functional category. The size of each rectangle is proportional to
the p-value for that category. d A set of 20 Magnaporthe oryzae-
unique genes upregulated regarding salt stress (25 min, 0.5 M KCl)
for which no homologues in other fungal species were found in public
databases
J Plant Dis Prot
123
best hits with the following cut-offs: E-value 1.0E-6 and an
internal annotation cut-off value of 55, to classify genes
into GO terms based on molecular function, biological
processes and cellular components. The results from the
BLASTp analysis, functional domain comparisons from
InterProScan (restricted to BlastProDom, HMMPfam and
TMHMM) implemented in Blast2GO and the prediction of
signal peptides with SignalP 4.1 software [15]; http://www.
cbs.dtu.dk/services/SignalP/), as well as interrogation
against the PHI database (version 4.0) were used to make
final assignments to GO functional categories of each gene,
indicated in the final table as ‘‘customized category’’.
Finally, GO enrichment (by focusing exclusively on the
biological process) of DE genes was computed using the
TopGO package (release version 2.26.0; [1] implemented
in Bioconductor 3.3, employing Fisher’s exact test for
statistical significance to extract the maximal meaning of
the RNA-Seq data. The enriched GO terms at p-values of
B 0.05 were retained and then slimmed in REVIGO [17];
http://revigo.irb.hr/) by removing redundant GO terms.
Treemaps were produced to visualize enriched and non-
redundant GO categories. A total of 130 of the ‘‘salt stress-
regulated’’ genes were not suitable for GO enrichment
analysis, because they represented hypothetical proteins or
displayed no identity to other proteins with assigned GO
terms in the database. The total list of DE genes obtained
from RNA-seq analysis, containing comprehensive infor-
mation regarding functional signatures for each gene, is
provided in Table S1. GO annotation obtained from JGI
(http://genome.jgi.doe.gov/Maggr1/Maggr1.home.html) is
also provided in Table S1 for augmented comparative
analysis.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://crea
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
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The exceptionality of stress response in : a set of “salt stress-induced” genes unique to the rice blast fungus

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