MoMyb1 is required for asexual development and tissue-specific infection in the rice blast fungus Magnaporthe oryzae
Dong et al. BMC Microbiology
MoMyb1 is required for asexual development and tissue-specific infection in the rice blast fungus Magnaporthe oryzae
Yanhan Dong 0
Qian Zhao 0
Xinyu Liu 0
Xiaofang Zhang 0
Zhongqiang Qi 0
Haifeng Zhang 0
Xiaobo Zheng 0
Zhengguang Zhang 0
0 Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education , Nanjing 210095 , China
Background: The Myb super-family of proteins contain a group of functionally diverse transcriptional activators found in plant, animal and fungus. Myb proteins are involved in cell proliferation, differentiation and apoptosis, and have crucial roles in telomeres. The purpose of this study was to characterize the biological function of Myb1 protein in the rice blast fungus Magnaporthe oryzae. Results: We identified the Saccharomyces cerevisiae BAS1 homolog MYB1 in M. oryzae, named MoMyb1. MoMyb1 encodes a protein of 322 amino acids and has two SANT domains and is well conserved in various organisms. Targeted gene deletion of MoMYB1 resulted in a significant reduction in vegetative growth and showed defects in conidiation and conidiophore development. Quantitative RT-PCR analysis revealed that the transcription levels of several conidiophore-related genes were apparently decreased in the Momyb1 mutant. Inoculation with mycelia mats displayed that the virulence of the Momyb1 mutant was not changed on rice leaves but was non-pathogenic on rice roots in comparison to the wild type Guy11. In addition, Momyb1 mutants showed increased resistance to osmotic stresses but more sensitive to cell wall stressor calcofluor white (CFW). Further analysis revealed that MoMyb1 has an important role in the cell wall biosynthesis pathway. Conclusion: This study provides the evidence that MoMyb1 is a key regulator involved in conidiogenesis, stress response, cell wall integrity and pathogenesis on rice roots in the filamentous phytopathogen M. oryzae.
Magnaporthe oryzae; Conidiogenesis; Stress response; Cell wall integrity; Pathogenesis
Rice blast caused by the heterothallic ascomycete
Magnaporthe oryzae, is the most destructive disease of
cultivated rice worldwide and can lead to severe losses of
annual rice yield [1,2]. Under normal conditions, the
fungus uses a highly specialized infection structure
appressorium generated from a conidium for plant
penetration [3,4]. After successful penetration, the invasive
hyphae grow rapidly in the host cells and caused blast
lesions. In 5 to 7 days, the pathogen produces numerous
conidia from the lesions and initiates a new infection
Regulation of gene expression at the level of
transcription controls many crucial biological processes. A
number of different factors, including transcription factors,
are essential for the process of transcription.
Transcription factors can recognize DNA in a sequence-specific
manner and modulate the frequency of initiation of
transcription upon binding to specific sites in the promoter
of target genes. The transcription factors can be
activators, repressors, or both usually display a modular
structure named the DNA-binding domain . In M. oryzae,
numerous transcription factors were identified and
characterized to be important for proper regulation of
infection related morphogenesis [6,7]. In our previous study,
many transcription factors, including MoCrz1, MoAp1,
MoAtf1, MoHac1, MoBzip10, MoSwi6 and MoMsn2
were reported to be involved in hyphal growth, asexual
development, stress response, infectious growth and
virulence by controlling the expression levels of a series
of target genes [8-13]. In plants, Myb protein family
comprises a large members of transcription factors .
The first identified MYB gene was the oncogene v-Myb
derived from the avian myeloblastosis virus .
Following v-Myb, a large and growing family of myb-related
genes were discovered in a wide variety of eukaryotes
including animals, plants, fungi and slime molds [16-18].
The Myb-related proteins contain a DNA-binding
domain and generally function in the regulation of cell
growth and differentiation, often by co-regulating gene
expression along with DNA-binding proteins of other
Myb proteins play important roles in controlling
phenylpropanoid metabolism, cell shape, and hormonal
responses during seed development and germination, and
cellular proliferation in plants . Additionally, two
Myb proteins from fungi, Cdc5 and flbD were also
reported to control cell shape [22,23]. In
Schizosaccharomyces pombe, the Cdc5p is essential for G2/M
progression and Cdc5 family members participate in a novel
pathway to regulate G2/M progression [22,24]. In
Aspergillus nidulans, flbD encodes a Myb-like DNA-binding
protein and is required for early conidiophore
development by activating a cascade of transcription factors for
conidiophore production [22,23]. Here, we investigate
the role of MoMYB1 in growth and infection-related
morphogenesis in M. oryzae. Deletion of MoMYB1
resulted in a failure to develop conidiophores and conidia,
and more tolerance to osmotic stressors. Furthermore,
MoMyb1 plays a crucial role in cell wall integrity and
tissue-specific infection of M. oryzae.
Fungal strains, cultures and transformation
The wild type strain Guy11, the mutants and the
complemented transformants were cultured on complete
medium (CM: 10 g D-glucose, 2 g peptone, 1 g yeast
extract, 1 g casamino acids, 50 ml 20 nitrate salts, 1 ml
trace elements, 1 ml vitamin solution, 15 g agar, add
distilled water to 1 L)  , straw decoction and corn
powder medium (SDC: 100 g straw, 40 g corn powder, 15 g
agar in 1 L distilled water), V8 juice agar medium
(100 ml V8 juice, 900 ml ddH2O, 0.2 g CaCO3, 15 g
agar) and oatmeal agar medium  at 28C. Protoplast
transformation was performed using hygromycin B
(HPH) and bleomycin as selective marker for gene
deletion and complementation assays as described .
Conidiation assays were performed as described .
Deletion and complementation of MoMYB1 in M. oryzae
The MoMYB1 gene deletion mutants were generated
using the standard one step gene replacement strategy
as described . The primer pairs FL4982/FL4983 and
FL4984/FL4985 (Additional file 1: Table S1) were used
to amplify the upstream and downstream flanking
sequence, respectively. The hygromycin resistance gene
cassette was prepared by primer pairs FL1111/FL1112
using pfu Taq DNA polymerase (TaKaRa) (Additional
file 1: Table S1). The hygromycin resistant transformants
were screened by genomic PCR, and further confirmed
by RT-PCR and southern blot analysis. For
complementation, the fragment containing the native promoter
region and the entire open reading frame (ORF) of
MoMYB1 were amplified by primer FL4841/FL4842
(Additional file 1: Table S1) and inserted into the pYF11
vector with a bleomycin resistance gene , and then
transformed into the Momyb1 mutant to obtain the
The two-week-old seedlings of susceptible rice cultivar
CO-39 were used to perform the detached leaf infection
assays. Mycelial plugs of the wild type Guy11, Momyb1
mutants and the complemented transformant were
inoculated on the intact leaves and kept in a moist chamber
at 28C for 24 h in darkness, followed by a 12/12 hour
light/dark cycle. Photographs were taken at 7 days after
inoculation. Root infection assays were performed as
described . Lesion formation was examined at 9 days
post-inoculation. The experiments were repeated three
times. For infectious hyphal growth on rice roots,
mycelia mats of Guy11 and Momyb1 expressing a GFP
protein were cultured in liquid CM medium at 28C for
2 days, then harvested and inoculated on the roots. After
48 h incubation under humid conditions at 28C, the
roots were observed under a fluorescence microscope.
Osmoregulation and CFW assays
Osmoregulation and CFW assays were performed as
described . Briefly, strain blocks were placed onto the
freshly prepared CM agar plates with NaCl (0.7 M), KCl
(0.6 M), and sorbitol (1 M), respectively, and cultured in
the dark at 28C for 7 days. For CM medium containing
cell wall perturbing agent Calcofluor White (CFW), the
final concentrations were 200, 400, and 600 g/ml of
CFW, respectively. The sensitivity was evaluated by
measuring the growth rate, and the experiments were
repeated three times with three replicates each time.
Cellular chitin content assay
Chitin (N-acetylglucosamine, GlcNAc) content was
determined as described [31,32]. Mycelia were freeze-dried
first. For each sample, 5 mg of dried biomass was
resuspended in 1 ml 6% KOH and heated at 80C for 90 min.
Samples were centrifuged and pellets washed with PBS
and resuspension. The pellets were finally resuspended
in 0.5 ml of McIlvaines buffer with Streptomyces plicatus
chitinase (Sigma, USA) and incubated for 16 hours at
37C with gentle mixing. 100 ml sample was then
combined with 100 ml of 0.27 M Mosadiumborate, heated
for 10 min at 100C, and 1 ml of freshly diluted (1:10) of
Ehrlichs reagent was added. After incubating at 37C for
20 min, 1 ml of the sample was transferred to a 2.5 ml
plastic cuvette (Greiner) and the absorbance at 585 nm
was recorded. Standard curves were prepared with
GlcNAc (Sigma, USA). The experiment was repeated
Protoplast release assay
The wild type Guy11 and the mutant strains were
cultured in liquid CM media for 2 days and the
mycelia were collected by centrifugation for 10 min at
5,000 rpm. The following lysis and protoplast release
steps were performed as described previously [33,34].
The mycelia were washed twice and resuspended using
20% sucrose. Lysing enzyme from Trichoderma harzianum
(Sigma-Aldrich, USA) was added to the suspension, with
lysis stopped after 30, 60, and 90 min. Protoplast release
were counted with a hemacytometer, and cell wall
degradation was examined with the light microscope. The
experiment was repeated three times.
Nucleic acid manipulation
DNA extraction and DNA gel blot hybridization were
performed using standard procedures as described .
Probe labeling, hybridization and detection were
preformed with the DIG High Prime DNA Labeling and
Detection Starter Kit (Roche Applied Science, Penzberg,
Quantitative RT-PCR assay
Two-week-old rice seedlings were inoculated with a
spore suspension of rice blast at 1 105 spores/ml. The
inoculated plants were placed in a chamber in the dark
for 24 h at 25C, and leaf tissues were collected at 8 h
and 48 h after inoculation. Mycelia were grown in liquid
complete medium for 48 h at 28C, 150 rpm and
harvested. Total RNA was extracted using the Invitrogen kit
as described previously .
For quantitative RT-PCR (qRT-PCR), 5 mg of total
RNA were reverse transcribed into first-strand cDNA
using the oligo (dT) primer and M-MLV Reverse
Transcriptase (Invitrogen). The qRT-PCR reactions were
performed following previously established procedures .
To compare the relative abundance of target gene
transcripts, the average threshold cycle (Ct) was normalized
to that of ACTIN gene (MGG_03982) as described .
P < 0.01 is used in the statistical test. The primer
pairs used in this section are listed in Additional file 1:
The full sequence of MoMYB1 was downloaded from
the M. oryzae online database (http://www.broadinstitute.
html) . Myb1 sequences from different organisms were
obtained from GeneBank (http://www.ncbi.nlm.nih.gov/
BLAST), using the BLAST algorithm . Sequence
alignments were performed using the Clustal_W 1.83
Identification of M. oryzae MYB1
The Myb family of proteins is a group of functionally
diverse transcriptional activators that is characterized by a
conserved DNA-binding domain of approximately 50
amino acids . Here, we identified an S. cerevisiae
BAS1 homolog MYB1 from the M. oryzae genome by a
BLAST_P search. MoMYB1 (MGG_06898.6) encoding a
protein of 322 amino acids possesses two SANT (a
putative DNA binding domain in the SWI-SNF and ADA
complexes, the transcriptional co-repressor N-CoR and
TFIIIB; InterPro: IPR001005) domains that are
interrupted by one intron . Alignment analysis showed
that MoMyb1 shares similarities to the homologs from
S. cerevisiae, A. nidulans, Schizosaccharomyces pombe,
Homo sapiens, Arabidopsis thaliana and Zea Mays
[22,23,41-45]. The amino acid sequence identity being
16%, 29%, 15%, 22%, 14%, and 21%, respectively. The
alignment of repeat DNA-binding domains was shown
in Additional file 2: Figure S1. Southern blot analysis
revealed MoMYB1 only had a unique copy in M. oryzae
genome (Additional file 3: Figure S2B)
MoMyb1 is highly expressed during conidia and plant
To gain insight into the functions of MoMyb1, we first
examined the gene expression profile at hyphal, conidial and
infectious stages of M. oryzae by qRT-PCR. In comparison
to the hyphal stage (1.0 0.1), the expression of MoMYB1
was significantly increased in conidial and infection stages.
The abundance of MoMYB1 was increased by 55-fold
(55.2 2.6) in conidial stage and increased by 40-fold
(40.4 8.9, 8 hours post inoculation: hpi) and 71-fold
(71.0 23.5, 48 hpi) in the infectious stage (Figure 1).
Targeted deletion of MoMYB1 in M. oryzae
To evaluate the role of MoMYB1 in growth and
development of M. oryzae, deletion mutant were generated by
replacing the MoMYB1 gene with the hygromycin
phosphotransferase resistance cassette (Additional file 3:
Figure S2A). Hygromycin resistant transformants were
first screened by genomic PCR and further confirmed by
southern blot analysis and RT-PCR (Additional file 3:
Figure S2B and S2C). Since all successful gene deletion
Figure 1 Expression profiles of MoMYB1 at different fungal
developmental stages. RNA was extracted from mycelia, conidia
and infectious stages (8 and 48 hpi), respectively. ACTIN was used for
normalization and values represent mean SD from two independent
experiments with three replicates each. Asterisks were indicated
significant differences at P < 0.01.
mutants yield the same phenotypes, only one mutant
was selected to analyze the biological phenotypes in this
study. For complementation, a 2.8 kb fragment
containing the native promoter and ORF of MoMYB1 gene
was cloned into pYF11  and reintroduced into the
Momyb1 mutant. The resulting transformants were
confirmed by RT-PCR to obtain the complemented strain
Momyb1/MoMYB1 (Additional file 3: Figure S2C).
MoMYB1 is involved in vegetative growth and is essential
To determine whether MoMYB1 was involved in growth
and conidiation, the wild type Guy11, MoMyb1
mutants and the complemented transformant MoMyb1/
MoMYB1 were inoculated on CM, SDC and V8 agar
plates. Compared to Guy11 and MoMyb1/MoMYB1,
the MoMyb1 mutants showed significant reduced
vegetative growth on these three media (Figure 2). The
conidiation was also quantified on CM, V8, oatmeal (OM) and
SDC media, the result revealed that the production of
conidia in the Momyb1 mutants was completely
abolished on these four media (Figure 3A). To find out the
potential reasons of the conidiation defect, we further stained
the aerial hyphe with lactophenol cotton blue as described
, and no conidiophores was observed in the MoMyb1
mutants, while normal gray conidiophores were formed in
the wild type Guy11 (Figure 3B).
MoMYB1 modulates the transcription of several
Because MoMyb1 is a putative Myb transcription factor,
which is required for an early stage of conidiation, we
speculate that MoMyb1 acts as a transcription factor
that regulates the expression of other conidiation-related
genes. To test this hypothesis, the expression of several
conidiation-related genes or their orthologs was analyzed
. The results revealed that MoMSN2, a homologue of
ScMSN2 of S. cerevisiae, and MoFLBC, homologous to
FLBC of A. nidulans, and glutamine synthetase (MoGLUS),
homologous to FLUG of A. nidulans, and MoSTUA,
homologous to STUA of A. nidulans, and MoCON8,
homologous to CON8 of N. crassa, was significantly
downregulated in the MoMyb1 mutant (Figure 4).
MoMYB1 plays an essential role in virulence on rice roots
but not on leaves
Since Momyb1 mutant have a conidiation defect, the
mycelial plugs of the wild type Guy11, Momyb1
mutant and the complemented transformant were
inoculated on the detached rice leaves to test the pathogenic
abilities. After 7 days incubation, the Momyb1 mutants
caused large and extended lesions similar to that of the
wild type Guy11 and the complemented transformant
(Figure 5A). We further examined the pathogenicity of
the Momyb1 mutant on rice roots. Unlike to the
results on rice leaves, the Momyb1 mutant caused no
virulence on roots 9 days after inoculation. In contrast,
the wild type Guy11 and the complemented
transformant caused typical rice blast lesions under the same
Figure 2 Colony morphology and vegetative growth of Guy11, Momyb1 and the complemented transformant (Momyb1/MoMYB1)
on complete media (CM), straw decoction and corn (SDC) and 10% V8 juice agar media (V8). Photographs were taken after 7-day incubation
in the dark at 28C. Asterisks indicated significant differences at P < 0.01. Values represent mean SD from three replicates each.
Figure 3 MoMYB1 is required for conidiophore development. (A) Conidia formation was observed under a light microscope 24 hours at
room temperature after induction of conidiation on cover slips. The strains were first grown on SDC media for 7 days. (B) Aerial cultures stained
with lactophenol cotton blue and observed under light microscope. Hyphae were stained blue, while conidiophores were in gray.
conditions (Figure 5B). These results suggested that
MoMyb1 played an important role in tissue-specific
infection of M. oryzae. To confirm this result, we observed
the infectious hyphal growth in the rice root by
transforming a green fluorescence protein (GFP) into wild
type Guy11 and Momyb1 mutant, respectively. The
results showed that the wild type could penetrate through
the root epidermis and formed branching invasive
hyphae at 48 h, while successful penetration and
development of invasive hyphae were rarely observed in the
Momyb1 mutant (Figure 5C and D).
Deletion of MoMYB1 results in more insensitive to salt
and osmotic stresses
To address the role of MoMYB1 in environmental
adaptation, the wild-type Guy11, Momyb1 mutant and the
Figure 4 MoMYB1 regulates the transcription of conidiation-related
homologous genes. RNA was extracted from mycelia cultured in
liquid CM medium at 28C for 2 days. ACTIN was used for
normalization, and the values were calculated by 2-ddCT methods
with quantitative RT-PCR data. Values represent mean SD from
two independent experiments with three replicates each. Asterisks
were indicated significant differences at P < 0.01.
Figure 5 Effect of MoMYB1 deletion on pathogenicity.
(A) Detached rice leaf assay. Intact rice leaves were inoculated by
mycelium plugs from wild type Guy11, Momyb1 mutant and
complemented transformant Momyb1/MoMYB1. Photographs were
taken at 7 days after inoculation. (B) Rice root infection assay.
Lesions were examined at 9 days post-inoculation. CK: inoculated
with agar plugs without hyphae. (C) Observation of the invasive
hyphal growth inside the rice root inoculated with the Guy11
and Momyb1 strain expression a GFP protein, respectively. White
arrows point to invasive hyphae. Bar = 50 m. (D) Close view the
invasive hyphae in plant cells. White arrows point to invasive
hyphae. Bar = 10 m.
complemented transformant were inoculated on the CM
agar plates containing the salt (0.7 M NaCl, 0.6 M KCl)
and osmotic (1 M sorbitol) stresses, respectively.
Compared with the wild-type and the complemented
transformant, the Momyb1 mutant showed less sensitivity
to NaCl, KCl and sorbitol (Figure 6A). The growth rate
of the Momyb1 mutant was much higher than that
of wild type and the complemented transformant
(Figure 6B). This result suggested that MoMYB1 has
a crucial role in response to salt and osmotic stresses.
Since Hog1 pathway was the most important signal
pathway to responsible for stress response in M. oryzae ,
we examined the expression of four major components
of the pathway, including MoSSK1, MoSSK2, MoPBS2
and MoOSM1 (MoHOG1). The results revealed that,
besides MoSSK1, the expression levels of MoSSK2, MoPBS2
and MoOSM1 were significantly decreased in the
Momyb1 mutant compared to the wild type Guy11
MoMyb1 is involved in cell wall integrity
To determine whether MoMYB1 has a role in
maintenance cell wall integrity, we test mycelial growth on CFW
which inhibit fungal cell wall assembly by binding
chitin. The results showed that the growth rate of the
Momyb1 mutant on CFW media was significantly
decreased, which reduced to 66.7%, 70.8% and 85.4% of
the wild type Guy11 under 200, 400, and 600 g/ml
CFW, respectively, while the complemented
transformant Momyb1/MoMYB1 can fully restore the defects
(Figure 7A and B). Chitin is a major component of
fungal cell wall and is synthesized by chitin synthases
and M. oryzae contains seven chitin synthases .
Therefore, we examined the transcription levels of these
chitin synthase genes by qRT-PCR analysis. The results
revealed that the expression of all chitin synthase genes
was significantly decreased in the Momyb1 mutant
(Figure 7C). We also examined the chitin content of the
mutant and found the chitin content was remarkably
reduced in the Momyb1 mutant compared to the wild
type (Figure 7D), indicating MoMYB1 has a role in cell
wall assembly. To further confirm this conclusion, the
mycelia of wild type Guy11 and the Momyb1 mutant
were treated with cell wall degrading enzyme. The results
showed that the Momyb1 mutant was more sensitive to
the enzyme treatment and released more protoplast after
incubation for 60 and 90 min compared to the wild type
Guy11 (Figure 8A). When observed at 60 min, the
Momyb1 mutant displayed a greater number of
protoplasts and no mycelia fragments were observed. In
contrast, the wild type showed much less protoplasts and
many mycelial fragments were found under the same
condition (Figure 8B).
In the present study, we characterized a Myb family
protein, MoMyb1 in M. oryzae and primarily focused on its
external phenotypes associated with pathogenesis.
Genetargeted replacement revealed that the loss of MoMYB1
led to a plethora of developmental defects in vegetative
Figure 6 Momyb1 mutants are more insensitive to osmotic stresses. (A) Wild type Guy11, Momyb1 mutants and complemented
transformant were incubated on CM plates containing various concentrations of NaCl, KCl or sorbitol at 28C for 7 days. (B) The growth rate was
determined 7 days after incubation at 28C by plotting the percentage of colonies in the presence of various concentrations of NaCl, KCl or
sorbitol against regular CM. (C) qRT-PCR analysis the transcription of four components of the Hog1 pathway in M. oryzae. Asterisks were indicated
significant differences at P < 0.01.
Figure 7 MoMyb1 has a role in cell wall integrity. (A) Wild type Guy11, Momyb1 mutant and the complemented transformant were
incubated on CM plate containing different concentrations of CFW at 28C for 7 days. (B) The growth rate was determined 7 days after
incubation at 28C by plotting the percentage of colonies in the presence of various concentrations of CFW against regular CM. (C) The
expression levels of seven chitin synthases encoding genes in the Momyb1 mutant. (D) GlcNa determination shows significantly decreased
chitin contents in the Momyb1 mutant. Data comprise three independent experiments with triple replications each time that yielded similar
results. Asterisks were indicated significant differences at P < 0.01.
Figure 8 Protoplast release of the wild type Guy11 and Momyb1 mutant. (A) Protoplast production of Guy11 and Momyb1 mutant
treated by cell wall degrading enzyme. Asterisks indicate a significant difference between the mutant and wild-type strain at P < 0.01. (B) Light
microscopic examination of protoplast release after treatment for 60 min and photographed.
growth, conidiation, stress response, cell wall integrity
Conidiogenesis and appressorium development are key
steps in the rice blast disease cycle. The fungus has
evolved regulatory networks to ensure the correct timing
and spatial pattern of these development events . The
expression profile of MoMYB1 at hyphal, conidial and
infectious stages of M. oryzae suggested that MoMyb1
might have a crucial role in conidial development and
plant infection. Deletion of MoMYB1 in M. oryzae
affected different developmental stages, including hyphal
growth, cell wall assembly and penetration. In addition,
the Momyb1 mutants displayed no conidia and
conidiophores, suggesting that deletion of MoMYB1 in M.
oryzae completely abolished conidiophore development and
thus affected conidium production. Therefore, we
conclude that MoMyb1 is a key regulator of M. oryzae for
conidium and conidiophore development. qRT-PCR
analysis revealed the expression level of five
conidiogenesisrelated genes were significantly decreased in the Momyb1
mutant, indicating that MoMyb1 probably functions as a
key upstream transcription factor in the conidiogenesis
signalling pathway to regulate the expression of genes which
involved in conidiophore and conidium development.
However, whether these conidiogenesis-related genes
directly regulated by MoMyb1 need further studies. One
surprise result was that the Momyb1 mutant caused rice
blast on rice roots, but not on leaves. Since the infectious
mechanism of M. oryzae on rice roots has been well
clarified , we conclude that the pathogenic difference in
Momyb1 on the two organs is a result of tissue-specific
infectious-related development mechanisms.
Cell fate specification is a process of fundamental
importance during development. In maize, GL1 encodes a
protein containing a Myb DNA-binding domain and is
expressed most highly very early during trichome
development and is a central regulator of the trichome cell
fate decision . Two Myb proteins from fungi, the
CDC5 gene product from S. pombe  and the FLBD
gene product from A. nidulans  can also control
aspects of cell shape, indicating Myb proteins are related
to cell wall integrity. Our results show that MoMyb1 is
involved in the response to multiple stresses via regulating
different signalling pathways including Hog1 pathway,
which contributes to osmoregulation. The insensitive or
hypersensitive of Momyb1 mutant to variety of stresses
may also indicate that MoMYB1 is involved in cell wall
Chitin is an integral part of the fungal cell wall and its
synthesis depends on the activity of chitin synthase
enzymes. In the present study, the Momyb1 deletion
mutants showed high sensitivity to the cell wall stressor
CFW. In M. oryzae, seven chitin synthases encoding
genes were characterized to involve in the development
and pathogenicity by affected chitin content . In
other phytopathogenic fungi, chitin synthases also play
crucial roles in proper regulation of infection-related
morphogenesis [50-54]. In the Momyb1 mutant, the
transcriptional levels of seven chitin synthases were
down-regulated and the chitin content was also
decreased in the mutant, suggesting that MoMyb1 may be
a multi-stress regulator of M. oryzae that contributes to
the loss of pathogenicity. MoMyb1 might directly or
indirectly regulate chitin synthesis, thus may disturb the
responses of cell wall signalling pathways to different
This study demonstrated that MoMyb1 functions as a
key regulator that important for vegetative growth,
conidiogenesis, stress response and pathogenicity in M.
oryzae. MoMyb1 is also involved in the maintenance of cell
wall integrity that are crucial for the growth and
development of the fungus.
Additional file 1: Table S1. Primers used in this study.
Additional file 2: Figure S1. Alignment of gene encoding repeats
domain among M. oryzae and other organisms. The predicted
amino-terminal sequence of MoMyb1 is compared with that of various
Myb-like proteins: Aspergillus nidulans FlbD ; Homo sapiens c-Myb
[41,42]; Arabidopsis thaliana Atr1 ; Zea Mays Cl ; Schizosaccharomyces
pombe Cdc5 ; S. cerevisiae Bas1 . The two repeating motifs of ~50
amino acids are grouped, and the amino acids identical in five or more are
showed in grey bars.
Additional file 3: Figure S2. Generation of MoMYB1 deletion mutant.
(A) Restriction map of the MoMYB1 genomic region. Disruption constructs
containing the homologous sequences flanking the HPH cassette to
replace MoMYB1. (B) Southern blot analysis of the wild type (lane 1, 2, 3
and 6) and Momyb1 mutant (lane 4, 5, 7 and 8) using MoMYB1 and HPH
specific probes, respectively. Genomic DNA was digested with EcoRI, SalI
and NdeI, respectively. (C) RT-PCR analysis of MoMYB1 in wild type,
Momyb1 mutants and the complemented transformant. The transcript
was lost in the Momyb1 mutants.
qRT-PCR: Quantitative reverse transcription polymerase chain reaction;
CFW: Calcofluor white; DNA: Desoxyribonucleic acid; ORF: Open reading
frame; BLAST: Basic local alignment search tool; SANT: a putative DNA
binding domain in the SWI-SNF and ADA complexes, the transcriptional
co-repressor N-CoR and TFIIIB.
YD, QZ, XL, X Zhang, ZQ, HZ and ZZ carried out the molecular genetic
studies, participated in the sequence alignment and drafted the manuscript.
YD and X Zhang participated in the sequence alignment. YD, QZ, HZ and ZZ
participated in the design of the study and performed the statistical analysis.
HZ, ZZ and X Zheng conceived of the study, and participated in its design
and coordination. All authors read and approved the final manuscript.
This research was supported by the National Science Foundation for
Distinguished Young Scholars of China (Grant No.31325022 to ZZ), Natural
Science Foundation of China (Grant No: 31271998, ZZ, Grant No: 31201471, HZ).
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